Biologics Bioanalalysis Compendium

Figure 2. LCMS of Peptides in Plasma. Nine synthetic peptides and their C13 stable isotope labeled counterparts were monitored in human plasma. Good separation was achieved between the peptides such that two acquisition time periods were used. Very good retention time stability was achieved and peak shapes on all peptides were of very good quality. Looped high resolution MS/MS scans (MRMHR scans) were developed to all 18 peptides.
Methods
Sample Preparation: Human plasma was depleted of the top
14 proteins using a MARS14 depletion cartridge. The sample was reduced, akylated, and digested, providing a solution of 0.9 μg/ μL plasma to act as the matrix for the experiment. Samples for the response curve were generated as previously described1,2, using the same set of peptides as in those studies. C12 versions of the peptides were spiked into plasma at varying amounts covering
the concentration range of 0.005 -250 fmol/uL, while C13 stable isotope labeled versions of the corresponding peptides were dosed at xed 5 fmol/uL level. For each sample, a 1μL injection was performed.
Chromatography: The sample was analyzed using the Eksigent nanoLC-Ultra® 2D System combined with the cHiPLC®-nano ex system in Trap-Elute mode. The cell lysate was loaded on the cHiPLC trap (200 μm x 500 μm ChromXP C18-CL, 3 μm, 300 Å) and washed for 10 mins at 4 μL/min. Then, an elution gradient of 5-35% acetonitrile (0.1% formic acid) in 45 mins was used on a nano cHiPLC column (75 μm x 15 cm ChromXP C18-CL, 3 μm, 300 Å). Trap and column were maintained at 30 oC for retention time stability.
Mass Spectrometry: Eluant from the column was sprayed using the NanoSpray® Source and heated interface into a TripleTOF® 5600 system (AB SCIEX). The acquisition method was set up with two acquisition time periods with 18 looped MS/MS (high
Figure 3. High Resolution Extracted Ion Chromatograms of Fragment Ions. In this experiment, MS/MS spectra were acquired in high sensitivity mode (resolution > 15000). To understand the effects of resolution on speci city for the fragment ions, post-acquisition XIC extraction was done at either 0.02 Da or 0.7 Da window width.
sensitivity mode) across them. Each had 200 msec accumulation time. The Q1 and CE were de ned for each targeted peptide and Unit resolution isolation was used on the Q1 quadrupole.
Data Processing: All data were processed using MultiQuantM Software 2.0. Fragment ion extractions were performed at 0.7 Da and 0.02 Da widths and the LLOQs were compared.
High Resolution MS/MS Spectra
Full scan MS/MS spectra can be acquired on the TripleTOF® 5600 System using either a high sensitivity (resolution > 15 000) or
high resolution mode (resolution >30 000). Depending on the speci city required, the data can also be extracted post-acquisition using variable width windows. In this experiment, looped
MS/MS spectra was acquired in high sensitivity mode, typical fragment ion peak resolutions of around 20 000 were observed. After acquisition, two different XIC widths were used on the same dataset, 0.7 Da to simulate what quadrupole resolution would look like, and 0.02 Da to investigate the effects of
higher resolution.
Easy Assay Development for MRMHR
When performing MRMHR quanti cation, full scan MS/MS is acquired all the time; so only the Q1 and collision energy must be determined upfront of collecting the dataset. It is during the post-acquisition data processing where the optimal fragment ions will be selected for use. Many fragment ions can be assessed after data collection for quantitative performance, such as lack of interference, best sensitivity, best combination for summing, etc.
As an example of interference assessment, multiple fragment ions for PSA peptide LSEPAELTDAVK were investigated for presence
of interferences by using the blank matrix injection. The internal standard heavy peptide is present in the blank and XICs to heavy peptide fragment ions can be used to determine the elution
time of the peptide (Figure 4, top). From here, the same elution
For Research Use Only. Not for use in diagnostic procedures.
20x
289.0948 133.0423
449.1903
Resolution – 22 800
y4
449.1902
1092.5103
1252.5404
964.4534
Resolution – 22 400
985.4558
963.0 964.0 965.0 966.0
m/z, Da
y5
612.2523
y7
863.4083 y8
200
100
100
0
747.3465
y6
715.3401
964.4531
y9
y10
0
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Time, min
500 1000
Mass/Charge, Da
www.absciex.com THERAPEUTIC PEPTIDE BIOANALYSIS
448.0 449.0 450.0 451.0
m/z, Da
51
Intensity
Intensity
MBP.YLASASTMDHAR
Myo.LFTGHPETLEK PSA.IVGGWECEK
CRP.ESDTSYVSLK
HRP.DTIVNELR HRP.SSDLVALSGGHTFGK
PSALSEPAELTDAVK APR.AGLCQTFVYGGCR
CRP.GYSIFSYATK
HIGH RESOLUTION ACCURATE MASS


HIGH RESOLUTION ACCURATE MASS
Intensity, cps Intensity, cps
Peptide
APR.AGLCQTFVYGGCR
APR.AGLCQTFVYGGCR APR.AGLCQTFVYGGCR APR.AGLCQTFVYGGCR APR.AGLCQTFVYGGCR
Fragment
y4, y5, y6, y7, y8, y9, y10
% CV
14.6
R2
0.9998
1500
1000
y8
MRMHR Quant – 0.02 Da XIC
19.93
y102+ y92+
y6 y8
LLOQ on column
25
% Accuracy
101
Heavy
Table 1. Advantage of Summing Fragment Ions. For peptide AGLCQTFVYGGCR, four different fragment ions were analyzed both individually and summed.
time for the light peptide fragment ion XICs can be analyzed (Figure 4, bottom). In this case, y8+, y9+ and y102+ fragments are determine to be free of interferences in the matrix blank, while the other three fragment ions show a small amount of interference at 20.1 mins that could impact the lower limits of quanti cation (LLOQ) for this peptide in matrix.
Again, because the underlying data is full scan MS/MS data, any number of individual fragment ions can be evaluated using
Light
100
19.03
50
0
20.09
20.0 20.5
50 100 50
5.5 8 19
500
150
97 97 98
0.9998 0.9996 0.9994
y5
y7
y8
y910013860.997 0
MultiQuantTM Software for quantitative performance on the
dataset. The standard concentration curves can be assessed to Figure 4. Easy Assay Development from Full Scan MS/MS Data. For the blank injec-
determine which fragment ions provide the best LLOQs (Table 1). Also, the fragment ions can be evaluated across the biological samples to determine which remain free of interferences across the sample set. This exible post-acquisition processing is only possible because of the full scan data.
When the noise is lower across a number of fragment ions, summing can sometimes improve observed LLOQs. Better ion statistics of summed signal can improve reproducibility and therefore LLOQs. Post-acquisition data processing decisions can be made because the full scan MS/MS data is always present.
This can be different for every peptide and every fragment ion, therefore needs to be determined during data processing through exploring the different combinations. Table 1 shows an example for the AGLCQTFVYGGCR peptide for Aprotinin protein.
Multiple fragment ions were evaluated for their individual sensitivity and then a subset were summed together. For this peptide, the summed case provides a slight improvement in the LLOQs obtained.
Impact of Fragment Ion Extraction Resolution on Speci city
In MRM analysis, both the Q1 and Q3 quadrupoles are set to transmit windows of ions of about 1Da wide. With MRMHR,
the fragment ions are analyzed as much higher resolution and therefore the post-acquisition XICs can be generated with very tight extraction windows of the user’s choice. To assess the impact of tighter resolution on speci city and sensitivity, the same dataset was analyzed using both wide 1 Da window extraction (to simulate MRM speci city) and narrow 0.02Da window extraction.
For one of the PSA peptides analyzed, the y7 ion is the most dominant fragment ion and therefore should provide the most
tion, XICs on fragment ions for both the heavy (top) and light peptides (bottom)
for PSA peptide LSEPAELTDAVK were generated. Heavy peptides were added as an internal standard and good signal at 19.9 min was observed. As there was no light peptide added in the blank, minimal signal should be observed. However, signal was observed in the y92+, y82+, y72+ ions for the light peptide indicating the presence of interferences that will impact the LLLOQs for this peptide. These fragment ions can now be avoided in the nal data processing.
sensitive quantitative data. However, the wide extraction analysis of this y7 ion (Figure 5 top) yielded a substantially worse 40 fold lower LLOQ than the narrow extraction case (Figure 5 bottom). Because the full scan data is stored, the MS/MS spectra can be investigated for the source of the interference. The full scan MS/MS at the point of peptide elution is shown in Figure 6 (bottom). Zooming in on the y7 ion m/z, it is clear that there is another fragment ion from another co-eluting peptide that has a very similar mass that cannot be resolved with quadrupole resolution (Figure 6 middle). However, if a very narrow extraction window is used, the y7 ion for PSA peptide can be selectively extracted to provide clean quanti cation data (Figure 6 top).
When there is little background or competing interferences, the high resolution extraction will not have a signi cant bene t. This is illustrated by the HRP peptide DTIVNELR (Figure 7). Using the de nition of LLOQ as better than 20% CV and between 80-120% accuracy, the LLOQs obtained for both the narrow and wide extraction windows are quite similar, 25 amol on column for the 07 Da extraction window and 50 amol on column for the 0.02 Da extraction window. Both peaks look very nice and the background noise is very low. The small improvement in LLOQ in the wider extraction case could be due to the fact that the entire fragment ion peak is extracted at this width providing improved ion statistics through more ion counts and therefore better reproducibility at the lowest level.
52 RUO-MKT-02-1486-A THERAPEUTIC PEPTIDE BIOANALYSIS www.absciex.com
19.5 20.0 20.5
19.5
Time, min
2+ y72+


Area Ratio
Area Ratio
HIGH RESOLUTION ACCURATE MASS
0.02 Da XIC
Calibration for IVGGWECEK.y7.Light: y = 1.31107 x + 0.68662 (r = 0.99963) (weighting: 1 / x)
R2 = 0.999 60 0.7 Da XIC
50 40 30 20 10
Calibration for IVGGWECEK.y7.Light: y = 1.40048 x + 0.01438 (r = 0.99820) (weighting: 1 / x)
70 R2 = 0.998
00 0 5 10 15 20 25 30 35 40 45
Concentration Ratio
Actual Conc
0 5 10 15 20 25 30 35 40 45 Concentration Ratio
10000.00 50000.00 250000.00
Actual Conc
5.00 10.00 25.00 50.00 100.00 250.00 500.00 1000.00 2500.00 10000.00 50000.00 250000.00
2 of 2 2 of 2 2 of 2
Num. V
0 of 1 0 of 1 0 of 3 0 of 3 0 of 3 0 of 3 0 of 3 0 of 3 0 of 3 2 of 2 2 of 2 2 of 2
1.149e4 4.821e4 2.503e5
Mean
N/A
N/A
N/A
N/A
N/A 2.367e2 5.392e2 1.028e3 2.490e3 9.826e3 4.476e4 2.553e5
Standard Devia
N/A
N/A
N/A
N/A
N/A 1.737e2 3.610e1 6.595e1 1.238e2 4.442e2 1.709e1 1.448e4
Percent CV
N/A N/A N/A N/A N/A 7.34 6.70 6.41 4.97 4.52 0.04 5.67
Accuracy Value #1
N/A 1.636e1 N/A N/A
N/A 3.681e0 N/A 2.977e1 N/A 1.190e2 94.69 1.740e2 107.84 4.977e2 102.82 1.055e3 99.60 2.407e3 98.26 9.512e4 89.53 4.478e4 102.13 2.480e5
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THERAPEUTIC PEPTIDE BIOANALYSIS 53
5.00 0 of 1 N/A
10.00 0 of 1 N/A 25.00 0 of 3 N/A 50.00 0 of 3 N/A 100.00 0 of 3 N/A 250.00 0 of 3 N/A 500.00 0 of 3 N/A 1000.00 0 of 3 N/A 2500.00 0 of 3 N/A
N/A N/A N/A 9.591e2
N/A N/A N/A 9.108e2 N/A N/A N/A 8.915e2 N/A N/A N/A 1.040e3 N/A N/A N/A 8.683e2 N/A N/A N/A 1.366e3 N/A N/A N/A 1.902e3 N/A N/A N/A 1.958e3 N/A N/A N/A 3.471e3 1.655e2 1.44 114.89 1.137e4 4.897e2 1.02 96.43 4.0787e4 3.303e3 1.32 100.12 2.480e5
60 50 40 30 20 10
0.7 Da XIC
Row Component Name
1 IVGGWECKy7.Light
2 IVGGWECKy7.Light 3 IVGGWECKy7.Light 4 IVGGWECKy7.Light 5 IVGGWECKy7.Light 6 IVGGWECKy7.Light 7 IVGGWECKy7.Light 8 IVGGWECKy7.Light 9 IVGGWECKy7.Light 10 IVGGWECKy7.Light 11 IVGGWECKy7.Light 12 IVGGWECKy7.Light
0.02 Da XIC
Row Component Name
1 IVGGWECKy7.Light 2 IVGGWECKy7.Light 3 IVGGWECKy7.Light 4 IVGGWECKy7.Light 5 IVGGWECKy7.Light 6 IVGGWECKy7.Light 7 IVGGWECKy7.Light 8 IVGGWECKy7.Light 9 IVGGWECKy7.Light 10 IVGGWECKy7.Light 11 IVGGWECKy7.Light 12 IVGGWECKy7.Light
Num. V Mean
Standard Devia
Percent CV
Accuracy
Value #1
Value #2
9.878e2 9.681e2 9.590e2 1.678e3 1.415e3 2.008e3 3.781e3 1.161e4 4.856e4 2.526e5
Value #2
2.330e1 4.390e1 3.321e1 2.244e2 5.566e2 1.076e3 2.431e3 1.014e4 4.475e4 2.451e5
Value #3
5.313e2 9.529e2 1.288e3 1.531e3 1.620e3 1.997e3 3.462e3
Value #3
N/A 3.606e1 1.221e2 2.490e2 5.633e2 9.531e2 2.632e3
Figure 5. Higher Resolution Fragment Extraction Can Improve LLOQ. For the PSA peptide IVGGWECEK, the most sensitive fragment ion is the y7 ion. The LLOQ for this pep- tide using a 0.02 Da extraction width was 250 amol on column. However, when a wider extraction window of 0.7 Da was used, the LLOQ was signi cantly impacted, only 10 fmol on column. Figure 6 illustrates at a spectral level this case.
For Research Use Only. Not for use in diagnostic procedures.


54
5fmC13, 0.9ug mtrx, 0.025fmC12 3-DTIVNELR_v6.Light (Standar... Area: 1.730e3, Height: 1.479e2, RT: 23.99 min
5fmC13, 0.9ug mtrx, 0.050fmC12 2-DTIVNELR_v6.Light (Standar... Area: 1.670e3, Height: 1.277e2, RT: 24.01 min
300 200 100
0
310.1768(1)
865.2 865.3 865.4 865.5 865.6
Mass/Charge, Da
Figure 6. Investigating MS/MS Interference for PSA Peptide IVGGWECEK. The full scan MS/MS is shown (Bottom). Zooming in on the y7 fragment ion (middle), the spectra from two different injections are overlaid. The blue trace is the injection
of 0.5 fmol of the C12 peptide on column, the pink trace is a more concentrated injection of 10 fmol on column. The top pane shows a further enlargement of the fragment ion. The peptide fragment of interest is the signal that increased from 0.5 fmol to 10 fmol. An extraction width of 0.7 obviously is not suf cient to resolve the target fragment from a nearly isobaric interference. An extraction width of 0.02 Da however, is suf cient and provides a clean XIC and a much improved LLOQ.
Conclusions
• Biological matrices are very complex and speci city is an important factor for assay robustness
• TripleTOF® 5600 System has the MS/MS sensitivity and speed to perform MRM-like analysis
• Post-acquisition extraction of fragment ions from the high resolution TOF MS/MS data allows for high speci city MRM-like data (MRMHR) to be obtained
• MRMHR can provide better quantitative detection limits in the presence of interferences and increase assay robustness
• Selecting best fragment ions or summing multiple fragment ions post- acquisition can improve detection limits
• Assay development is simpli ed as fragment ion selection is post- acquisition
References
1. Keshishian H et al. (2007) Molecular and Cellular Proteomics, 6, 2212-2229. 2. Addona TA, et al. (2009) Nature Biotechnology, 27, 633 – 641.
For Research Use Only. Not for use in diagnostic procedures.
© 2010 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEXTM is being used under license. Publication number: 2780411-01
Peptide
0.7 Da
300 200 100
0
0.02 Da
Interference
2400 2000 1600 1200
800 400 0
864.0 864.5 865.0 865.5 866.0 866.5
Mass/Charge, Da
00 23.0 23.5 24.0 24.5 25.0
Time, min
23.0 23.5 24.0 24.5 25.0
Time, min
233.1291(1)
677.8365(2) 759.3689(2)
532.9292(3) 553.7768
200 400 600 800 1000 1200
Mass/Charge, Da
969.4881(1)
Figure 7. Measuring the Impact of Extracted Ion Width on LLOQ for Peptide DTIVNELR. For the wider extraction window of 0.7 Da, the standard curve was linear down to 25 amol on column (left). The background noise was minimal in this example, so when the higher resolution extraction was performed the LLOQ results were very similar (50 amol, right).
130 120 110 100
90 80 70 60 50 40 30 20 10
24.01
140 120 100
80 60 40 20
23.99
25 amol 0.7 Da XIC 13.3% CV
50 amol 0.02 Da XIC 10.7% CV
RUO-MKT-02-1486-A THERAPEUTIC PEPTIDE BIOANALYSIS www.absciex.com HIGH RESOLUTION ACCURATE MASS
Intensity, cps
Intensity, cps
Intensity, cps
Intensity
Intensity


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For Research Use Only. Not for use in diagnostic procedures.
55
HIGH RESOLUTION ACCURATE MASS


56
Application of Differential Ion Mobility Mass Spectrometry to Peptide Quantitation
Using SelexIONTM Differential Mobility Separation Technology for better selectivity for peptides in complex mixtures on the AB SCIEX Triple QuadTM 5500 LC/MS/MS System
TTA Zerr, L Meunier, SW Wood, P Struwe
Celerion Switzerland AG, 8320 Fehraltorf, Switzerland
Key scienti c challenges of peptide quant assays
Reduced recovery, low sensitivity – The adsorptive properties and/or polarity of peptides can compromise recovery, and interferences from biological matrices can negatively impact sensitivity and selectivity.
Limited quantitation range – Poor MS/MS sensitivity combined with often poor selectivity can compromise the desired lower limits of quantitation (LLOQ).
Limited MRM selectivity – MRM approaches and ef cient UHPLC separations may not provide adequate signal-to-noise ratios at LLOQ due to isobaric interferences or high baseline noise.
Key bene ts of differential mobility separation (DMS) for peptide quant assays
Background noise reduction enhances LLOQs – For cases where background noise limits LOQ, DMS provides an additional level of selectivity, orthogonal to the mass spectrometer and
LC system.
Better sensitivity even with less re ned sample prep
– Selectivity gains from DMS permit less selective sample preparations, allowing for overall improvements in sensitivity due to more ef cient extractions and better recovery.
Selectivity improvements overcome sensitivity losses – DMS is often accompanied by a loss in absolute sensitivity, but the gains in selectivity improve the potential for real gains in LLOQ.
Key features of SelexIONTM differential mobility separation technology for peptide quant assays
Separation of diverse species reduces baseline noise – SelexION Technology separates isobaric species, and single and multiple charge state interferences to reduce background levels and achieve better selectivity and LOQs –while retaining compatibility with UHPLC/MRM speeds.
Simple installation and maintenance – DMS is truly orthogonal to LC and MSMS; installs in minutes with no tools required and no need to break vacuum. Device maintenance is minimal and very straightforward.
Differential Mobility Cell
Compact and simple design allows the cell to be installed without the use of any tools and in less than two minutes.
SelexIONTM Curtain Plate
Updated version of the traditional curtain plate to accommodate the differential ion mobility cell. Maintains the same level of robustness and stability associated with the original design.
Shortened assay cycle – SelexIONTM Technology can potentially reduce chromatographic runtimes.
Ef cient separation process – Planar geometry results in short residence times, high speeds, and minimal diffusion losses for maximum sensitivity and UHPLC compatibility.
Chemical modi ers for further selectivity – Introducing chemical modi ers to the homogenous elds of the SelexIONTM Device cell allows for ampli cation of the separation capacity and adds a new dimension of selectivity.
RUO-MKT-02-1486-A THERAPEUTIC PEPTIDE BIOANALYSIS www.absciex.com
ORTHOGONAL SELECTIVITY TOOLS


Compatible with high-throughput, regulated environments
M
– SelexION Technology provides ruggedness and stability to
enable high performance quantitative bioanalysis under GLP settings.
Introduction
The quantitative determination of therapeutic peptides to
support pharmacokinetic and toxicokinetic studies can sometimes be challenging. Poor MS/MS sensitivity combined with poor selectivity fragments can compromise the desired lower limits of quantitation (LLOQ). In addition, the adsorptive properties and/or polarity of peptides can compromise recovery, and interferences from biological matrices can negatively impact sensitivity and selectivity. In such cases, MRM approaches – even when combined with ef cient UPLC separations – may not be suf cient to provide adequate signal-to-noise ratios at LLOQ in the presence of isobaric interferences or high baseline noise.
Differential ion mobility spectrometry (DMS) may provide a useful tool in such instances by providing an additional, orthogonal degree of selectivity. Although DMS analysis is often accompanied by a loss in absolute sensitivity, the gains in selectivity may be suf cient enough to realize real gains in the LLOQ. Alternatively, selectivity gains from DMS may permit a less selective sample preparation to be used, while still delivering overall improvements in sensitivity due to improved extraction recovery.
This poster presents two case studies were the SelexIONM Differential Mobility Separation Device was evaluated for the quantitation of therapeutic peptides. Selectivity, sensitivity and precision of peptide measurements obtained in experiments with and without DMS were compared. The potential bene ts and limitations of the technique are discussed.
Figure 1: Optimization of separation voltage. The optimization of SV is performed by constant infusion at low analyte ow in solution whilst ramping the compensation voltage.
Materials and methods
Sample preparation
Celerion proprietary peptides, A and B, were used for all experiments.
For Research Use Only. Not for use in diagnostic procedures.
Chromatography
Peptide A-LC system: Pumps:
Auto sampler: Column:
Column temperature: Injection:
Flow rate:
Mobile phase A: Mobile phase B: Gradient:
Series 200 Micro Pump from Perkin Elmer Pal CTC from CTC Analytics
Onyx monolithic C18 100x3 mm
Room temperature
20 uL
0.8 mL/min
Methanol/Water/Formic acid 5:95:3 v/v/v Methanol/Water/Formic acid 95:5:0.2 v/v/v
Time (min) 0
0.5 1.0 2.0 4.0 7.0 7.5
%A %B 100 0 100 0
35 65 25 75 10 90
0 100 100 0
Peptide B-LC system: Pumps:
Auto sampler:
Column temperature: Injection:
Flow rate:
Mobile phase A: Mobile phase B: Gradient:
Acquity Binary Solvent manager from Waters
Acquity Sample Manager/Organizer from Waters Column Ascentis Peptide ES C18 50x2.1 mm 2.7 um
Room temperature
20 uL
0.3 mL/min
0.02% Acetic acid aqueous Methanol
Time (min) %A %B 0 90 10 1.0 90 10
3.0 5 95
3.1 90 10
4.0 90 10
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THERAPEUTIC PEPTIDE BIOANALYSIS
57
ORTHOGONAL SELECTIVITY TOOLS


58
Table 1: MS/MS, MRM, and SelexIONTM Device parameters for peptides A and B on the AB SCIEX QTRAP® 5500 System. SelexIONTM device settings
Figure 2: Optimization of compensation voltage. As compensation voltage (COV)
is in uenced by mobile phase and source conditions, on column COV optimization is performed by injection of analyte at a ow rate and mobile phase composition comparable to the intended LC conditions. In some instances, additional selectivity may be achieved by use of a modi er (e.g., methanol, acetonitrile, isopropanol, acetone) introduced into the SelexIONTM Device at low ow. No modi er was used in the case studies presented.
The SelexION Device needs only a few minutes for installation onto the AB SCIEX Triple QuadTM 5500 LC/MS/MS System and can be accomplished without breaking the MS vacuum. For the best performance, equilibrating the electrode for 20–30 min at the desired temperature (low/med/high) is required.
Within the device, ions are separated by differential mobility
due to an individual molecule’s size and shape. An optimized combination of separation voltage (SV) and compensation voltage (COV) separates the analyte from background ions.
Optimization of these parameters is very simple and can be performed as part of instrument tuning. The optimization of SV
is performed by constant infusion at low analyte ow in solution whilst ramping COV (Figure 1). The optimal combination of separation and compensation voltages gives the most separation whilst maintaining maximum peak intensity. Although optimal SV is usually obtained at around 4500 V, a lower value can be chosen to ensure system robustness and stability. As COV is in uenced by mobile phase and source conditions, on column COV optimization is performed by injection of analyte at a ow rate and mobile phase composition comparable to the intended LC conditions (Figure 2).
Data processing
Samples were acquired with the Analyst® 1.5.2 Software. Quanti cation was completed with MultiQuantTM Software.
RUO-MKT-02-1486-A THERAPEUTIC PEPTIDE BIOANALYSIS www.absciex.com MS/MS Settings
Peptide A Peptide B
Ion source/polarity ESI/Positive ESI/Positive
CAD High High
CUR 30 30
TEM 700 °C 700 °C
Gas 1 70 50
Gas 2 50 60
Ion Spray Voltage 5500 V 5000 V
MS/MS Settings
Peptide A Labeled IS for A Peptide B Labeled IS for B
Transitions 1029.3/136.0 1106.7/123.0 656.4/249.0 661.4/249.0
Dwell Time (msec) 150 100 100 100
Resolution Q1/Q2 Unit Unit Unit Unit
SelexIONTM Device Settings
Peptide A Peptide B
DT (temperature) Low High
DR (throttle gas) Off Off
COV 11.5 15.0
DMO -3 -3
SV 3500 3500
Spiked level of peptide A (ng/mL)
8 20 50 100
Precision (CV%)
without SelexIONTM N/AP 25.0 13.7 6.0
Device
Precision (CV%)
with SelexIONTM 6.3 7.8 8.1 6.3
Device
n6666
Table 2: Lower limit of quantitation (LLOQ) data for peptide A.
ORTHOGONAL SELECTIVITY TOOLS


Figure 3: Peptide A at 20 ng/mL from a protein precipitation extract from human plasma without DMS.
Figure 5: Peptide B at 0.04 ng/mL extracted with solid phase extraction without DMS.
Figure 7: Peptide B at 0.08 ng/mL extracted with protein precipitation without DMS.
Figure 4: Peptide A at 20 ng/mL from a protein precipitation extract from human plasma with DMS.
Figure 6: Peptide B at 0.04 ng/mL extracted with solid phase extraction with DMS.
Figure 8: Peptide B at 0.08 ng/mL extracted with protein precipitation with DMS.
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For Research Use Only. Not for use in diagnostic procedures.
59
ORTHOGONAL SELECTIVITY TOOLS


60
Accuracy (%)
Precision (%)
Concentration (ng/mL)
+ DMS
- DMS
+ DMS
- DMS
0.04
100
100
10.2
7.4
0.08
101
101
6.0
4.8
0.2
97.7
97.6
4.7
2.2
0.8
97.0
97.2
2.4
0.9
1.6
102
102
2.2
0.6
10
103
103
1.3
1.2
Linear regression r value
0.9976
0.9987
Table 3: Linearity, accuracy, and precision for peptide B peak area measurements with and without DMS. Intra-run replicates, n=6, for SPE extracts with and without DMS. Concentration curves were analyzed with linear t and a 1/x2 weighting.
Results and discussion
Case study 1
In this case study, a proprietary peptide (peptide A, MW 4,113.7 g/mol) was evaluated. This peptide is known to exhibit adsorptive characteristics, and solid phase extraction (SPE) cleanup (reverse phase or mixed mode) from human plasma results in very low recoveries. As a consequence, protein precipitation with methanol was the only feasible extraction approach from human plasma. Additionally, due to poor fragmentation, a wide selection of fragments was not available for quantitation, and fragment
m/z 136 was the only suitable fragment displaying adequate sensitivity. Under these conditions, the resulting LLOQ is severely compromised by the lack of selectivity, despite separation using a 6 min chromatographic gradient (Figure 3). Without DMS, using MRM with +ESI (Table 1), an LLOQ of only 50 ng/mL is achievable from human plasma. An elevated baseline and a number of closely-eluting peaks were observed for spectra obtained for peptide A, which required careful set-up of peak integration parameters.
Using the same protein precipitation procedure and LC gradient conditions, DMS was added to the work ow (optimized parameters, Table 1), and peptide A spectra were evaluated for any improvement in selectivity. A signi cant improvement was observed. Despite a loss of absolute signal (approximately a factor of 5), a reduction of background interference of approximately
a factor of 20 was observed. This resulted in an overall gain in S/N of an approximate factor of 4-5 (Figure 4). This facilitated a lowering of the feasible LLOQ from 50 to 8 ng/mL (Table 2) without changing extraction or gradient LC conditions.
Case study 2
In this case study the quantitation of a therapeutic peptide (peptide B, MW 1,311 g/mol) in rat plasma was evaluated. This peptide was extracted from rat plasma using polymer-based, reversed-phase SPE. A recovery of 65% was achieved. Samples were chromatographed on a fused-core peptide column using a methanol/water gradient with formic acid as the acid modi er.
Using +ESI-MRM (Table 1), a range of 0.04–10 ng/mL could be routinely achieved. At LLOQ a S/N of 10 (analyte intensity 1,000 counts, background 100 counts) resulted in a precision of 7.4% (Figure 5). Applying DMS to this method resulted in approximately a 10-fold decrease in background with absolute analyte sensitivity exhibiting approximately a 6-fold decrease. Whilst S/N improved to 16 at LLOQ (0.04 ng/mL), there was no marked improvement in precision at this level (Table 3). A lowering of LLOQ could also not be facilitated under these conditions. Background, however, was almost completely eliminated allowing for easier and more consistent peak integration (Figure 6).
A protein-precipitation approach was also evaluated for peptide
B. As absolute recovery was compromised using SPE, it was anticipated that a lowering of the LLOQ by a factor of 2 could be achieved by combining recovery gains of protein precipitation with selectivity gains from DMS. Without the bene ts of SPE cleanup
in this instance, an LLOQ of only 0.08 ng/mL could be achieved without DMS due to high background and closely eluting isobaric interferences (Figure 7). With DMS, all background interferences were removed (Figure 8). However, due to the inherent loss of absolute signal associated with DMS, the resulting LLOQ achieved was limited to the LLOQ demonstrated for SPE extraction.
Conclusions
Differential ion mobility spectrometry provides a useful additional or orthogonal selectivity during the quantitation of peptides (and conventional small molecules). For applications involving peptide quantitation in particular, selectivity gains may be signi cant as separation of multiply-charged analyte precursors from singly- charged background interference. The true gain in sensitivity as a function of absolute sensitivity and selectivity will be analyte dependant and will also be in uenced by choice of MRM transition, chromatographic separation and extract cleanliness
Often for peptides sensitivity is already compromised by a number of factors including low bioavailability, adsorption to surfaces, formation of multiple charge states and poor or non selective fragmentation. In these instances additional tools to aid lowering of LLOQs are to be welcomed. In some cases, true gains in sensitivity may not be realised or required, but improvements in selectivity may bring other bene ts – namely, simpler extraction methods, shorter chromatographic runs, or improved peak integration.
References
1 A. Zerr, L.Meunier, S.Wood, P. Struwe. Application of Differential Ion Mobility Mass Spectrometry to Peptide Quantitation. Nov 14–16 2012. Celerion Poster Presentation. EBF 2012 Open Symposium, Barcelona.
RUO-MKT-02-1486-A THERAPEUTIC PEPTIDE BIOANALYSIS www.absciex.com ORTHOGONAL SELECTIVITY TOOLS


For Research Use Only. Not for use in diagnostic procedures.
www.absciex.com THERAPEUTIC PEPTIDE BIOANALYSIS
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ORTHOGONAL SELECTIVITY TOOLS


62
Improving Intact Peptide Quantitation with
Differential Mobility Separation and Mass
Spectrometry (DMS-MS)
Using SelexIONTM Technology coupled with the QTRAP® 5500 System for additional selectivity and separation
TTTof peptides in biological matrices
J. Larry Campbell and J.C. Yves Le Blanc
AB SCIEX, 71 Four Valley Drive, Concord, ON, L4K 4V8 Canada
Key challenges of large peptide quantitation
Poorly fragmenting peptides – Cyclic fragments often fragment poorly resulting in few product ions for analysis.
Challenging Physico-chemical proprieties of peptides such as non-speci c binding, poor solubility, complex charge state envelope makes peptide quanti cation makes peptide quanti cation challenging.
Sub pg level sensitivity – Need of very low levels of quantitation (pg/mL range) and lack of target functional groups for sample clean-up.
Key bene ts of SelexIONTM Technology for peptide quantitation
SelexIONTM Technology (differential mobility spectrometry) separates isobaric species, single and multiple charge state interferences and reduces background levels to achieve better selectivity and thereby LOQs – all while being compatible with UPLC/MRM speeds.
SelexIONTM Technology is the only ion mobility technology with the ruggedness and stability to enable high performance quantitative bioanalysis under GLP settings.
SelexIONTM Technology is truly orthogonal to LC and MS/MS with no tools required and no need to break vacuum to install.
The QTRAP 6500 System enables MRM3 capabilities that provide an order of magnitude of selectivity improvement over standard MRM techniques and can enable faster separations without
the need for long LC run times to eliminate/reduce background interference.
Key features of SelexION
Chemical Modi ers – The introduction of chemical modi er adds a new dimension to selectivity and dramatically increases separation capacity.
Planar geometry results in high speed and minimal diffusion losses for maximum sensitivity and UHPLC compatibility.
(A)
Extension ring Curtain DMS Orifice for coupling of plate cell plate TurboV Source
(B)
Figure 1: (A) Components associated with SelexIONTM Technology mounted on 5500 QTRAP® System. (B) Cross section of the DMS cell showing the introduction of the chemical modi er, typically set at 1.5% (or higher) of the total curtain gas ow supplied. Cell dimensions are 1 x 10 x 30mm (gap height x width x length).
RUO-MKT-02-1486-A THERAPEUTIC PEPTIDE BIOANALYSIS www.absciex.com
ORTHOGONAL SELECTIVITY TOOLS


Highly robust, reproducible, and stable for use in regulated bioanalysis.
Easy to maintain, and can be Installed or removed in minutes with no need to break vacuum or use any tools.
Introduction
Differential mobility spectrometry (DMS) can be used to separate isobaric species and co-eluting interferences by ltering selected compounds based upon the difference in the ions’ mobilities during the high- and low- eld periods of the applied asymmetric waveform. This waveform, termed the separation voltage (SV)
is applied across the gap between the two planar electrodes. Applying a compensation voltage (CoV) ensures the selective transmission of a particular species through the DMS device
at a speci c SV, while the other, unwanted chemical species
in the mixture are ltered out. Peptides tend to have CoVs
that are several volts higher than singly-charged ions, and this characteristic provides a way to easily discriminate amongst multiply-charged peptides when using DMS, allowing for detection, with some degree of selectivity, using the single ion monitoring (SIM) mode. Furthermore, the addition of a chemical modi er into the transporter gas signi cantly alters CoVs, increasing the capacity of DMS to separate these molecules [1-2].
During the drug discovery and development process, it is necessary to separate and quantify peptides from a biological matrix with a high degree of selectivity and sensitivity. For peptides that do not fragment well (such as cyclic peptides) or ones that over-fragment (resulting in too many product ions), analysis by SIM is essential, but often confounded by high background noise and low sensitivity. To overcome these challenges, we applied
AB SCIEX SelexIONM Technology, a planar differential mobility separation device, to a mixture of intact peptides and explored how DMS can selectively lter peptides from chemical noise. In this current work, we show that DMS mass spectrometry provides gas phase separation of peptides, while reducing background noise and co-eluting interferences, improving the selectivity of peptide detection in a manner comparable to MRM detection.
Materials and methods
Sample preparation
Two matrices were used to generate chemical noise: 1) protein- precipitated plasma produced using perchloric acid (HClO4)
and 2) trypsin-digested plasma. For HClO4-precipitated plasma, plasma (1 mL) was vortexed with 5% HClO4 (v:v) for 5 min.
After centrifugation (16,500 g, 10 min.), the resulting supernatant was mixed with 100 μL of 10% ammonium hydroxide (v:v). The precipitated plasma was diluted 2-fold prior to injection on column. Digested plasma was generated according to conventional protocols (overnight trypsin incubation). To eliminate undigested large proteins post-incubation, plasma was precipitated with acetonitrile (1:1 v:v) and diluted 2-fold prior to injection on column. Angiotensin I, angiotensin II, angiotensin
IV, neurotensin, dynorphin A, melittin and exenatide were spiked
at different concentration levels in both matrices. Exenatide was obtained from ChemPep (Wellington, FL), and all other peptides were obtained from Sigma Aldrich (St. Louis, MO) and used without further puri cation.
Chromatography
LC System: Column: Injection: Flow rate: Mobile phase:
Gradient:
Mass spectrometry
System:
System interface:
Ion source gas 1 (GS1): Ion source gas 2 (GS2): Curtain gas (CUR): Temperature (TEM): IonSpray voltage Floating (ISVF):
Scan type: Accumulation time: Declustering potential: Collision energy: Collision energy spread:
For Research Use Only. Not for use in diagnostic procedures.
LC system Poros (1 x 10mm, 7 μM)
1 μL ( xed loop) 175 μL/min
A) water, 0.5% formic acid
B) acetonitrile, 0.5% formic acid
Time/min A% B% 0 98 2 0.5 98 2
5 75 25
6 20 80 7.5 20 80
AB SCIEX QTRAP® 5500 System
Turbo VM Source and the reduced volume electrospray ionization (ESI) probe (65μm ID) for peak dispersion minimization.
30
60
20 500 °C
+5500 V
Single-ion monitoring (SIM) sec for each scan experiment
10 V
0 V
M Eksigent ExpressHT
www.absciex.com THERAPEUTIC PEPTIDE BIOANALYSIS
SIM conditions were used for all peptides. Retention time (RT) and CoV values are listed in Table 1. SIM was used to evaluate the additional selectivity that would originate from the DMS cell.
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ORTHOGONAL SELECTIVITY TOOLS


64
DMS cell conditions
SelexIONTM Technology, a planar DMS device, attaches between the curtain plate and ori ce plate of the QTRAP 5500 System (Figure 1A). Drawn towards the ori ce by the curtain gas as it ows to the MS ori ce, ions uctuate between the at plates when an asymmetric waveform is applied. An ion’s net drift is based on
the difference in the ion’s mobility in the high eld (K(E)) and low eld mobility (K(0)) conditions of the applied waveform (Table 1). A compensation voltage (CoV), a small DC offset between the plates, is applied as a ltering voltage – and must be optimized for each ion for speci c analytical conditions. Isopropanol (IPA), acetonitrile (ACN), and methanol (MeOH) can be used as chemical modi ers and introduced into the transport gas via the curtain gas (Figure 1B), prior to an ion’s entrance into the DMS device, thereby altering the separation characteristics of analytes [1-2]. However, this feature was not evaluated in the present study. The SV and the associated CoV were tuned for each analyte. The DMS can also be used in transparent mode, whereby the SV and CoV voltages are turned off, allowing the MS system to operate in conventional mode (as if the DMS device was not installed).
Results and discussion
Elimination of co-eluting, multiply-charged interferences using SelexIONTM Technology
Table 1 lists the compensation voltages (CoV) obtained for all peptides under LC conditions. Several peptides have optimum CoVs in the +10.0 to +11.5 V range (typically the highest charge state) or unique CoV values can be found for some of charge states. In many cases, the charge state associated with these unique CoVs is not necessarily the highest possible charge state, but is for the charge state with the highest response. Figure 2 shows the distribution of the optimum CoV observed for each peptide versus the peptide’s m/z – and includes values obtained
for several singly-charged compounds at similar separation voltages, aptly demonstrating that singly-charged species (including peptide-based) can be discriminated from the multiply- charged signals.
To investigate the extent of background noise reduction when using DMS, individual peptides were spiked into protein- precipitated or digested plasma and were monitored in SIM mode.
Figure 3 shows the chromatogram obtained for angiotensin I, II and IV when monitored in SIM mode. In the presence of both matrices, DMS data showed signi cant baseline reduction over data collected in transparent mode (to mimic a non-DMS- ltered triple quadrupole environment). Peptides spiked into perchloric
Figure 2: Distribution of observed CoV values as a function of mass-to-charge. Charge states are color coded. A separation voltage (SV) of 3500 V was used. Included are values for non-peptide singly-charged species under similar conditions.
HCIO4 Precipitated Plasma Digest Plasma
DMS ON DMS OFF DMS ON DMS OFF
Angio I
Angio II
Angio IV
Figure 3: Detection of angiotensin I, II and IV in SIM mode under optimized DMS conditions and DMS in transparent mode (mimic regular MS system) in different matrices. For angiotensin 1, z=+4, +3 and +2 are represented by blue, pink and orange trace, respectively. For angiotensin II and IV, z=+3, +2 and +2 are represented by the blue, pink and orange traces, respectively.
RUO-MKT-02-1486-A THERAPEUTIC PEPTIDE BIOANALYSIS www.absciex.com
ORTHOGONAL SELECTIVITY TOOLS


Figure 4: Detection of angiotensin I, II and IV in SIM mode under optimized DMS conditions and DMS in transparent mode (to mimic a non-DMS environment) in different matrices. For angiotensin 1, z = +4, +3 and +2 are represented by blue, pink and orange trace, respectively. For angiotensin II and IV, z = +3, +2 and +2 are represented by the blue, pink and orange traces, respectively.
Figure 5: Selectivity of melittin spiked in digested plasma at different levels when detected by LC-DMS-SIM. Comparing DMS at optimum values (DMS ON) versus DMS operated in transparent mode (DMS OFF). Concentration are in fmol/μL and chromatograms are for the melittin z = +5 ion.
www.absciex.com THERAPEUTIC PEPTIDE BIOANALYSIS
DMS Transparent Mode
DMS Optimized (SV 3400)
For Research Use Only. Not for use in diagnostic procedures.
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ORTHOGONAL SELECTIVITY TOOLS


66
To increase the separation capacity of the DMS, a chemical modi er (e.g., 2-propanol) can be added in the curtain gas (Figure 1B) [1-2]. Preliminary results using chemical modi ers such as 2-propanol (IPA) and acetone for the analysis of intact peptides have not signi cantly improved the separation capability of the DMS cell (data not shown). In general, the peptide CoVs shift towards a lower-value charge state after exposure to the chemical modi er, but the observed shift (∆CoV) remains the same for all charge states for a given peptide. Occasionally , the increased proton af nity of the chemical modi er can lead to proton abstraction from select peptide charge states, resulting in signal loss. This phenomena can, in some cases, assist in further reducing chemical interference but can also lead to additional tuning requirements (monitoring of additional charge states). This concept will be explored in future work, as well as the effects of adding af nity labels (e.g., SCIEX iTRAQ® Reagents) to further enhance peptide separation by DMS.
Conclusions
Differential mobility spectrometry (DMS) using SelexIONTM Technology separated peptides from co-eluting interferences, improving selectivity in a complex biological background by reducing background noise.
Signi cant baseline reduction was observed for angiotensin I, angiotensin II, and angiotensin IV spiked into plasma when ltering peptides using DMS.
Selectivity for melittin in plasma was improved by DMS, with chromatograms showing fewer interfering peaks and diminishing background noise, even over a range of peptide concentrations (0–100 fmol/μL).
References
1 Schneider, B.B.; Covey, T.R.; Coy, S.L.; Krylov, V.E.; Nazarov, E.G., Anal.Chem. 2010 (82) 1867-1880
2 Schneider, B.B.; Covey, T.R.; Coy, S.L.; Krylov, V.E.; Nazarov, E.G, ThOC am 08:50, Proceedings of 58nd ASMS Conference on Mass Spectrometry and Allied Topics, Salt Lake City, May 23-27 (2010)
Figure 6: Calibration curve for melittin spiked in digested plasma and monitored in SIM mode for the Z=+5 ion. Duplicate injections were made and a Wagner t was used.
acid-precipitated plasma were selectively detected using SIM. For all peptides ltered by DMS, both the noise level and extra LC peaks were eliminated from the resulting chromatograms, showing very low baselines for both matrices (Figure 3).
For samples analyzed in digested plasma, most noise in the baseline was reduced for all peptides, except for the z = +2 ion of angiotensin II, which displayed additional LC peaks that were unresolved from the analyte peak (Figure 3). When compared
to data collected in transmission mode for angiotensin II, the DMS- ltered data showed a signi cant reduction in chemical interferences. Because of the high fragmentation ef ciency of the angiotensin II z = +2 ion, the combination of DMS with MS/MS detection could increase selectivity for this peptide; however, it is likely that selectivity gains by MS/MS detection would be offset by the loss of signal incurred by high levels of secondary peptide fragmentation during MS/MS.
The fragmentation characteristics of peptides can signi cantly impact the sensitivity of their detection. Many intact peptides, such as melittin, do not fragment easily and generate low ion counts; DMS ltering is expected to improve the sensitivity of low-fragmenting peptides due to a lessening of chemical noise and the increased selectivity inherent in the DMS method. Chromatograms obtained for the +6, +5 and +4 charge state
of melittin spiked in digested plasma show a signi cant drop in baseline noise and elimination of additional peaks for the +6 and +5 charge states when ltered with DMS (in SIM mode) and resulted in no additional loss of signal (Figure 4). The enhancement of selectivity by DMS improved melittin detection in complex matrices, and required minimal tuning of DMS parameters (SV/CoV). Overall, DMS improves the selectivity of melittin in a concentration-dependent manner, as demonstrated by heightened analyte peaks over baseline when compared to chromatograms obtained without DMS (Figure 5.)
TTTRUO-MKT-02-1486-A THERAPEUTIC PEPTIDE BIOANALYSIS
www.absciex.com
TORTHOGONAL SELECTIVITY TOOLS


PUSHING THE LIMITS IN SELECTIVITY
For Research Use Only. Not for use in diagnostic procedures.
AB SCIEX SelexIONM Technology
A NEW DIMENSION IN SELECTIVITY
Ion mobility spectrometry for quantitative and qualitative applications
SelexIONM technology on the AB SCIEX Triple QuadM 6500 and QTRAP® 6500 systems delivers a new dimension of selectivity and performance for any application requiring the separation of isobaric species, isolation of challenging co-eluting contaminants and reduction of high background noise.
Explore a new dimension at www.absciex.com/selexion
© 2014 AB SCIEX. For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of AB SCIEX Pte. Ltd. or their respective owners. AB SCIEXM is being used under license.


68
Differential Mobility Separation Mass
Spectrometry for Quantitation of Large
Peptides in Biological Matrices
Using SelexIONTM Technology Coupled with QTRAP® 5500 System
Derek T. Wachtel, Sanjeev T. Forsyth & Robert W. Busby Ironwood Pharmaceuticals, Cambridge, MA
Key challenges of large peptide quantitation
Poorly fragmenting peptides – Cyclic fragments often fragment poorly resulting in few product ions for analysis.
Challenging Physico-chemical proprieties of peptides such as non- speci c binding, poor solubility, complex charge state envelope makes peptide quanti cation very challenging.
Sub pg level sensitivity – Need of very low levels of quantitation
(pg/mL range) and lack of target functional groups for sample clean-up.
Key bene ts of SelexIONTM Technology and the QTRAP® 5500 System for large peptide quantitation
Separation based on analyte structure – SelexIONTM Technology is a planar differential mobility separation device (DMS) that separates peptides based on difference in their chemical and structural properties.1
More options for selectivity – SelexIONTM Technology adds an orthogonal level of separation and selectivity prior to the instrument ori ce (Figure 1).1
Compatible with high performance bioanalysis – SelexIONTM Technology is compatible with fast cycle times required for monitoring multiple MRM transitions combined with narrow HPLC peaks.
Key features of SelexIONTM
Chemical Modi ers – The introduction of chemical modi er adds a new dimension to selectivity and dramatically increases separation capacity.
Planar geometry results in high speed and minimal diffusion losses for maximum sensitivity and UHPLC compatibility.
Highly robust, reproducible, and stable for use in regulated bioanalysis.
Derek Watchel
TTEasy to maintain, and can be Installed or removed in minutes
RUO-MKT-02-1486-A THERAPEUTIC PEPTIDE BIOANALYSIS www.absciex.com
with no need to break vacuum or use any tools.
Introduction
The ability to quantify large peptides in a biological matrix with adequate selectivity and sensitivity depends on several factors:
1) the presence of multiple charge states, 2) varied fragmentation ef ciency due to size and structure, and 3) the complexity
of the matrix to cause varied background and interferences. Tryptic digestion can reduce the size of the peptide and improve fragmentation, but this process introduces an additional step, which is undesirable in a high-throughput environment.
Detection limits for MRM acquisition can be heavily affected by the fragmentation characteristics of the peptide. Sensitivity is reduced if the peptide resists fragmentation or if it fragments too extensively (spreading the ion current across many product ions). Cyclic peptides are especially dif cult to detect because they tend to have poor MS/MS ef ciency. For these cases, high sensitivity can sometimes be achieved by monitoring the intact
TTORTHOGONAL SELECTIVITY TOOLS


additional level of selectivity to LC/MS/MS providing gas phase separation of isobaric species and co-eluting interferences to reduce background noise. Here, the utility of a SIM work ow combined with DMS for speci city was investigated for quantifying large therapeutic peptides in a protein-precipitated plasma matrix.
Materials and methods
Sample preparation
To extract the peptides from plasma, an aliquot of each standard (100 μL) was mixed with 400 μL of 5% formic acid in acetonitrile (with 150 ng/mL peptide). After vortexing for 10 min, the sample was centrifuged at room temperature for 10 min (21,000 x g). The supernatant (425 μL) was transferred to a clean 96-well plate and evaporated to dryness at 50 oC. The sample was reconstituted with acetonitrile (30 μL) and shaken for 15 min. After mixing with 2.5% formic acid in water (20 μL), the sample was shaken for an additional 15 min.
Figure 1: High selectivity quanti cation using SelexIONM Technology on the QTRAP® 5500 System. SelexIONM Technology1 is an easy-to-install differential mobility device available on a QTRAP® 5500 or 6500 System that is used to provide additional selectivity to any quantitative experiment. An asymmetric waveform alternates between high eld and low eld, and ions will have a net drift towards one of the plates based on their high and low eld mobility difference. A compensation voltage (CoV) is applied as the ltering voltage, which is tuned for the compound of interest. Other co-eluting isobaric or non- isobaric species tune with different CoVs and will be ltered away.
peptide in single ion monitoring (SIM) mode. However, SIM methods are not routinely utilized due to the resulting reduced selectivity and high background levels from monitoring only the parent ion without fragmentation.
Differential mobility separation (DMS) mass spectrometry adds an
Figure 2: Multiple reaction monitoring (MRM) scans of peptide PN1944 using the QTRAP® 5500 System. Multiple reaction monitoring (MRM) scans provide a high degree of selectivity for detecting peptides in complex matrices. However, detection of some large peptides is limited by their fragmentation pattern, which ultimately limits the sensitivity of the MRM mode – either the peptide does not fragment well, or the peptide fragments into many product ions. The LLOQ (125 ng/mL) achieved in rat plasma using this approach was not suf cient to achieve detection limits required for the experiment.
Source Curtain DMS extension plate cell
ring
For Research Use Only. Not for use in diagnostic procedures.
To maximize signal intensity for each peptide, the following optimal parameters were used:
DMS temp: low Modi er: none Separation voltage: 3800 Compensation voltage: 15.5 DMO offset: -5.5 DMS resolution → off
Chromatography
LC system: Column:
Column temp.: Injection:
Flow rate: Mobile phase:
Gradient:
Mass spectrometry
System: Interface:
DMS parameters
Acquity UPLC System (Waters)
Thermo-Hypersil Gold (2.1 x 50 mm, 1.9 μm)
40 °C
5 μL
800 μL/min
A) water, 0.1% formic acid
B) acetonitrile, 0.1% formic acid
Time/min A% B% 0 95 5 0.5 95 5
3 5 95
QTRAP® 5500 System
SelexIONM Technology device and Turbo VM Source
www.absciex.com THERAPEUTIC PEPTIDE BIOANALYSIS
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ORTHOGONAL SELECTIVITY TOOLS


70
Analysis is done using a single ion monitoring (SIM) acquisition strategy. Single ion monitoring (SIM) is a variation of an MRM experiment where the parent ion is monitored in Q1 and Q3 without fragmentation in Q2. For large peptides, SIM methods can provide larger signal without the need for extensive compound optimization.
Data processing
Results were analyzed using Analyst® Software 1.5 quanti cation tools.
Results and discussion
High-throughput peptide quantitation
Optimizing the extraction method and chromatographic conditions for every test compound being evaluated is not desirable for high- throughput analyses. To overcome the challenges of evaluating peptide levels in complex biological matrices, a simple extraction method and high-throughput chromatographic conditions were developed and optimized for selectivity and sensitivity for a diverse set of peptides (~30 amino acids, 4 kDa) in multiple lots of rat plasma prior to analysis by LC/MS/MS.
Time-saving, generic MS acquisition methods are attractive in
a high-throughput environment, but these methods are often limited by background noise, delivering reduced sensitivity and limiting the ability to generate a suitable pharmacokinetic (PK) pro le for a peptide. MRM methods are the gold standard for high sensitivity quanti cation, but occasionally, the fragmentation properties of large peptides can limit the ultimate sensitivity achieved (Figure 2).
Monitoring the intact form of the peptide using SIM can provide better sensitivity (Figure 3, top), but the resulting spectra have much higher background noise that further impacts detection
limits. Combining the higher intensity of SIM with the added selectivity of differential mobility separation (DMS) can provide the necessary selectivity for improving detection limits. Compared to those achieved with SIM alone, the signal-to-noise ratio shows a signi cant improvement when using DMS to detect peptides due to the substantial reduction of baseline noise (Figure 3, bottom).
Assay Performance using DMS with SIM
The lower limit of quanti cation (LLOQ) for the DMS + SIM acquisition strategy was 4 ng/mL (Figure 4, top), approximately
a 30-fold improvement over the LLOQ from conventional MRM scans (125 ng/mL, Figure 2) . The statistics for the peaks observed at 4 ng/mL are shown in the table (Figure 4); good reproducibility and accuracy were achieved.
TTFigure 3: Signal-to-noise ratio improves using DMS. (Top) Poor selectivity is observed during the analysis of peptide PN1944 in SIM mode (monitoring precursor m/z in Q1 and Q3 with no collision energy for higher sensitivity). The peptide signal at 16 ng/ mL is largely obscured by background noise. (Bottom) Analysis of the same sample with DMS shows greatly improved signal-to-noise for the target peptide.
RUO-MKT-02-1486-A THERAPEUTIC PEPTIDE BIOANALYSIS www.absciex.com
ORTHOGONAL SELECTIVITY TOOLS


Conclusions
Differential Mobility Separation (DMS) using SelexIONM Technology provides an orthogonal level of selectivity by separating components based on their chemical properties and ion mobility.
Matrix interferences and background noise can be signi cantly reduced to improve selectivity and thus sensitivity.
An LLOQ of 4 ng/mL for a 30 amino acid peptide (~4 KDa) was achieved in protein-precipitated rat plasma, providing an over 30-fold improvement in the MRM analysis strategy.
The DMS + SIM acquisition strategy combined with a high-throughput sample preparation and LC work ow achieves the desired LLOQ (4 ng/mL) for PK studies of large therapeutic peptides.
References
1 AB SCIEX SelexIONM Technology: A New Solution to Selectivity Challenges in Quantitative Bioanalysis – Differential Mobility Separations Enhanced with Chemical Modi ers: A New Dimension in Selectivity. AB SCIEX Technical Note, Publication 2960211-01.
2 Multiple Mass Spectrometric Strategies for High Selectivity Quanti cation of Proteins and
For Research Use Only. Not for use in diagnostic procedures.
Peptides: Solve the Most Challenging Quantitative Problems with QTRAP Technical Note, Publication 5310212-01.
®
5500 System. AB SCIEX
Theoretical Concentration (ng/mL)
Replicate
Sample
Measured Concentration (ng/mL)
Relative Error (%)
Average Relative Error (%)
CV (%)
4
1
1
3.5
-12.5
2.2
13.5
2
4.2
5
3
4.84
21
4
5.08
27
2
1
4.17
4.3
2
4.25
6.3
3
4.35
8.7
4
4.3
7.5
3
1
3.86
-3.5
2
3.15
-21.3
3
3.61
-9.8
4
3.75
-6.3
www.absciex.com
THERAPEUTIC PEPTIDE BIOANALYSIS
Figure 4: Lower limit of quantitation for a 4 kDa peptide in plasma using the DMS+SIM high-throughput quanti cation work ow. (Top) The chromatograms for the blank plasma and the LLOQ using the DMS + SIM approach are shown. (Bottom) The statistics for the measurements at the LLOQ are shown. Using the DMS+SIM strategy provides an ~30-fold improvement in detection limit over the conventional MRM approach.
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72
UGT Family of Enzymes: Quanti cation of
Tryptic Peptides using SelexIONTM Technology
TTT®
on the QTRAP 6500 System
Part 3 of 3: Using SelexIONTM Technology on the QTRAP 6500 LC/MS/MS System for additional selectivity
by separating multiple charge-state ions in tryptic digests
Suma Ramagiri, Loren Olson, Gary Impey, Carmen Fernandez Metzler
AB SCIEX, 71 Four Valley Drive, Concord, ON, L4K 4V8 Canada Pharma Cadence,
Key challenges of peptide selectivity
Co-eluting multiple charge interference limits accurate quanti cation and also peak integration at LOQ levels.
Isobaric interferences will limit selectivity and speci city of the assay and cause issues for accurate identi cation during bioanalytical method development process.
Key bene ts of SelexIONTM Technology and the 6500 Series for tryptic peptide quantitation
Background or interfering ions can be tuned out prior to any MS ltering. This leads to a boost in performance through several avenues; LOQ improvement, reduction in background or interfering ions, isobaric separation or an accelerated runtime.
Whether facing the challenge of resolving a chromatographic interference, eliminating a high baseline, or separating isomers, AB SCIEX SelexION Technology offers a powerful new tool to help the bioanalytical scientist solve tough selectivity challenges.
Key features of SelexION
SelexIONTM Technology (differential mobility) separates isobaric species, single and multiple charge state interferences and reduces background levels to achieve better selectivity and thereby LOQ’s – all while compatible with UPLC/MRM speeds.
Excellent inter-day and intra-day precision and accuracy can be achieved with SelexION Technology, to satisfy bioanalytical validation requirements.
DMS is truly orthogonal to LC and MSMS with no tools required and no need to break vacuum to install and provide ruggedness and stability to enable high performance quantitative bioanalysis under GLP settings.
SelexIONTM Technology provides the potential to reduce chromatographic runtimes.
Carmen Fernandez
TTRUO-MKT-02-1486-A THERAPEUTIC PEPTIDE BIOANALYSIS
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TORTHOGONAL SELECTIVITY TOOLS


Introduction
In this tech-note series on improving peptide quantitation, we again use the UDP-glucuronosyltransferase (UGT) enzyme family to highlight an additional selectivity option for the complex analysis of peptides in biological matrices. We previously discussed the importance of UGT enzymes in glucuronidation which have a major role in phase II metabolism – arguably the most important human pathway for elimination of the top 200 drugs. Our studies focus on the quantitation of very low-levels of UGT peptides, providing absolute protein concentrations used to calculate the necessary pharmacokinetic parameters and dosing schedules for drug trials.
LC/MSMS technologies enable highly precise and accurate quantitation of the UGT enzymes, but require advanced analytical performance to reach desirable LOQs. In Part 1, we introduced the 6500 series of instruments that provide up to 5-fold improvement in the LOQ over other MS systems – using the standard MRM approach and micro ow LC strategies to assist with UGT peptide detection in the low nanogram/mL level. Then, in Part 2, we investigated how MRM3 scans can offer improved selectivity through a fragmentation pathway speci c to the ion of interest – to help eliminate closely, and, even, co-eluting interferences.
As researchers encounter more complex samples, the separation performance and LOQs must be enhanced through a variety of methods. Improvements to standard MRM approaches are limited to adjustments in ion pairs, selection of different CE pro les, modi cations in sample preparation and/or chromatography
to enhance the signal on analytes of interest. Often these adjustments are very time-consuming and don’t provide signi cant improvement over the original methodology. QTRAP Technology offers MRM3 capabilities, providing an additional selectivity option for those analytes with a generous spectrum of fragment ions. If the fragment ions demonstrate an inadequate signal for further quantitation or are not speci c enough to be advantageous, there is another truly orthogonal technique available on both the QTRAP and triple quadrupole systems – SelexIONM Technology.
In this third and nal installment, we highlight a selectivity approach that uses SelexIONM Technology to take advantage of an analyte’s differential mobility in high and low energy elds for an additional level of separation in LC/MS/MS work ows.
Materials and methods
Sample preparation
Standard tryptic digestion procedures were applied to 10 lots of human liver microsomes. Standard curves were made
using digested rat liver microsomes and BD Supersome Human UGT products.
Chromatography
LC system: Column:
Column temp.: Injection:
Flow rate: Mobile phase:
Eksigent ekspert microLC 200
Eksigent ChromXPM 3C18-300-CL (1.0 x 50 mm, 3 μm, 300 Å)
40 °C
5 μL
50 μL/min μL
A) water, 0.1% formic acid
B) acetonitrile, 0.1% formic acid
For Research Use Only. Not for use in diagnostic procedures.
Gradient: Time/min A% B% 0 100 0 0.5 100 0
5 75 25
6 20 80 7.5 20 80
Mass spectrometry
System: Interface:
Data processing
QTRAP® 6500 System
SelexIONM Technology device in positive MRM mode
The MRM transitions were. These transitions were determined to be optimal based on MS/MS analysis (not shown). Data were quantitated using MultiQuantM Software.
Source extension ring
Curtain DMS plate cell
DMS Dimensions 1x10x30 mm
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THERAPEUTIC PEPTIDE BIOANALYSIS
Figure 1: SelexIONM Technology (Differential Mobility Separation (DMS): The DMS
cell is easily mounted without any tools, and consists of two planar plates that direct ionized sample in the gas ow laterally towards the MS ori ce. An asymmetric waveform is applied which alternates between high eld and low energy elds, resulting in a separation voltage (SV). As the ions move back and forth between the plates, they are dragged towards the exit with the gas ow and will have a net drift towards one of the electrodes based on their high and low eld mobility. A small
DC offset, the compensation voltage (CoV), is applied to one electrode and must be optimized to ensure transmission of the ion of interest through the cell. The COV can be considered a ltering voltage, and tuning individual peptides at speci c COV values separates the peptide from unwanted background and interfering peaks.
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74
Enhanced product ion (EPI)
10X increase in signal intensity
EPI scan with Q0 Trapping
DMS OFF
Elimination of multiple charge state background noise
EPI scan with Q0 Trapping
DMS ON
Figure 2: The QTRAP® 6500 System offers multiple options to enhance sensitivity, while still acquiring qualitative/sequence information. In this case Q0 trapping was leveraged to boost sensitivity, while a looped EPI scan provided full scan MS/MS sequence data at UHPLC speeds. The resulting chimeric spectrum – resulting from a co- eluting interference – would have taken much longer to identify using conventional triple quadrupole technology. SelexIONTM Technology was then leveraged to remove the interference, yielding a clean MS/MS spectrum for quantitation.
TIDA
Multiple charge state interferences
EPI with Q0 Trapping
DMS to purify spectra (COV=9C)
No interferences
Interference Peak IEIYPTSLTK
Results and discussion
Elimination of co-eluting multiple charge interference using SelexIONTM Technology
When optimizing MRM transitions for quantitation of
low-level tryptic peptides from tissues samples, co-eluting interferences can occasionally contaminate peptide MS/MS spectra making sequence con rmation dif cult, as observed with the analysis of the IEIYPTSLTK peptide of UGT isoform
2B7 (UGT2B7). Using the QTRAP 6500 System coupled with the MIDASTM Work ow, the MS/MS spectra initially indicated that only y ions were present, along with many additional, unexplainable peaks (Figure 2). The QTRAP MIDASTM Work ow
is a single-injection, targeted work ow only available on the QTRAP Series of instruments, where MRM transitions are used as survey scans to quantify the key peptides in complex samples. By employing Q3 as a fully functional ion trap, the QTRAP System simultaneously identi es and con rms the identity of peptides
by capturing full scan MS/MS spectra. An alternative approach using looped enhanced product ion (EPI) scans with Q0 trapping produced a much stronger signal, revealing a chimeric MS/MS spectra resulting from a closely-eluting contaminant. To eliminate the contaminating signals and provide a pure MS/MS spectra of IEIYPTSLTK, SelexIONTM Technology was used to lter the peptide of interest form the background. The EPI scan is the linear ion trap version of a regular product ion scan, but the response is more intense (by orders of magnitude) because the ions of interest are
collected and stored in the trap. As a result, the EPI scan produces a high quality MS/MS spectrum for a speci c peptide fragment. The fragmentation occurring in the collision cell provides the information-rich MS/MS spectrum typical of collisionally-activated dissociation fragmentation. The additional information gained from the b ions discovered in the cleaner spectrum, con rmed the identity of the IEIYPTSLTK peptide.
Reduction in LC analysis time while sustaining selectivity with SelexIONTM Technology
Coupling SelexIONTM Technology to the 6500 series platforms enables exploration of a new dimension in selectivity prior to MRM analysis. Along with the reduction of background noise and removal of interfering ions, SelexIONTM Technology provides the additional bene t of shortening the chromatographic runtime. Extensive sample preparation prior to chromatography can be reduced as well by SelexIONTM Technology because of the elimination of multiple charge-state ions – which may interfere with the detection of selected peptides in complex matrices (e.g., tryptic digests). Selected ion monitoring (SIM) assays for poorly- fragmenting peptides (e.g., cyclic peptides) are also feasible with SelexIONTM Technology.
A deeper look at how SelexIONTM Technology separates multiple charge states?
The DMS offers a degree of selectivity that is dependent on the chemical nature of the analyte. For intact peptides, the multiply
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ORTHOGONAL SELECTIVITY TOOLS


charged ions (Z > +2) tend to optimize at elevated compensation voltages (CoVs). As a consequence of the higher CoVs, the ion of interest is moved into a CoV space that is often free of other interfering ions. Figure 3 illustrates the separation of multiple charge states from a mixture of BSA/Myoglobin/ß-Gal digests.
Conclusions
SelexION Technology takes advantage of the mobility differences that ions experience as they travel through a set of plates with high and low elds applied. Background or interfering ions can be tuned out prior to any MS ltering. This leads to a boost
in performance through several avenues; LOQ improvement, reduction in background or interfering ions, isobaric separation or an accelerated runtime.
The spectrum for EIYPTSLTK peptide of UGT isoform 2B7 was deconvoluted when interfering ions were removed by SelexION Technology, allowing for further sequence con rmation and quantitation for this peptide.
Multiple charge states can be separated from each other due to the increase in CoV relative to the number of charges per ion, allowing for a mixture of isobaric, multiply charged species to be separated.
References
1 Fernández-Metzler C. (August 2013) “Peptide Quanti cation on the QTRAP® Mass Spectrometers with Micro owLC: Bridging the Best of Small Molecule and Proteomic Analysis.” AB SCIEX
Mass Spec Webinar Series. Retrieved at: http://www.absciex.com/events/webinars/peptide- quanti cation-on-the-qtrap-mass-spectrometers-with-micro owlc--bridging-the-best-of-small- molecule-and-proteomics-analyses.
1“UGT Family of Enzymes: Quanti cation of Tryptic Peptides. Part 1 of 3: The QTRAP® 6500 Platform and MicroLC Provide the Combination of Sensitivity, Speci city and Robustness for the Quantitation of UGT Enzymes.” (White Paper) AB SCIEX. Accessed November 2013. Retrieved at: www.absciex.com
1 UGT Family of Enzymes: Quanti cation of Tryptic Peptides. Part 2 of 3: Accelerating MRM3 Work ows on QTRAP® 6500 System for Enhanced Selectivity in Complex Matrices like Tryptic Digests.” (White Paper) AB SCIEX. Accessed November 2013. Retrieved at: www.absciex.com
1 UGT Family of Enzymes: Quanti cation of Tryptic Peptides. Part 3 of 3: Using SelexIONM Technology for Additional Selectivity by Separating Multiple Charge State Ions in Tryptic Digests.” (White Paper) AB SCIEX. Accessed November 2013. Retrieved at: www.absciex.com
For Research Use Only. Not for use in diagnostic procedures.
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76
MRM3 Quantitation for Highest Selectivity in Complex Matrices
TAB SCIEX QTRAP® 5500 System Innovations
Mass spectrometry has transformed quantitative analysis to become the method of choice for many assays. More recently, LC/MS/MS has revolutionized quantitative bioanalysis. While single MS ltering offers advantages over non-mass selective techniques, the use of tandem mass spectrometry (MS/MS, or MS2) eliminates interferences and results in a dramatic increase in selectivity which yields a very low baseline, excellent limits of quanti cation, and very good linearity. As a result, the Multiple Reaction Monitoring (MRM) experiment performed on triple quadrupole mass spectrometers has become the technique of choice for highly sensitive and selective quanti cation in biological matrices.
In some cases, interferences cannot be eliminated using MRM. More elaborate sample cleanup and chromatography is required to eliminate these interferences. If a high baseline or matrix interference cannot be eliminated, the result is a compromised Lower Limit of Quanti cation (LOQ) as the detection of compounds in complex matrices is limited by signal-to-noise rather than by raw instrument response. In such cases, the addition of a third MS stage has been shown to greatly increase selectivity and eliminate the high baseline or chromatographic interference. The result is a lower LOQ and better chromatographic peak shape.
Sensitive LC MS/MS/MS analysis requires instrumentation with three key performance features: the ability to perform MS3, ultra high sensitivity to overcome the loss in ion current due to
multiple fragmentation steps, and fast scan speeds to keep up with fast LC. These requirements have been dif cult to achieve with results comparable to MRM mode until the development of the AB SCIEX QTRAP® 5500 LC/MS/MS System.
Key Features of the QTRAP® 5500 System for MRM3 Quanti cation
MRM3 quanti cation – On the QTRAP® 5500 System, an MS3 scan is performed with a fast cycle time and using a narrow scan range centered around the second generation product ion to be used for quanti cation. This type of scan is referred to as MRM3 (Figure 1).
Faster linear ion trap scan speeds – Scan speeds up to 20,000 Da/sec enable MS3 scans with an HPLC compatible cycle time such that extracted ion chromatograms (XICs) of second generation product ions can be extracted and integrated with a suf cient
number of data points across the chromatographic peak.
Better in-trap fragmentation – The new Linear AcceleratorTM Trap with pulsed gas valve implemented in the QTRAP® 5500 System provides faster, more ef cient in-trap fragmentation (Figure 2).
Highest available ion trap sensitivity – The QTRAP® 5500 System features the highest sensitivity commercial ion trap mass analyzer.
High selectivity – Unit isolation of precursor ion in Q1 followed by excitation and fragmentation at unit resolution in the ion
trap provides the highest available selectivity in MRM3 analysis (Figure 3).
Speed
The speed and ef ciency of ion-trap fragmentation has also been greatly improved on the QTRAP® 5500 System. Collision gas is now introduced through a high speed pulsed gas valve that enables a rapid increase in pressure in the LIT (Figure 2). Together with an increase in the RF drive frequency, this results in increased fragmentation ef ciency and reduced excitation time of 25 ms or less.
In addition, the scan speed of the linear ion trap has been increased to a maximum of 20,000 through the use of the faster eQTM electronics. This enabling fast MRM3 scan cycles at highest sensitivity.
Selectivity
The combination of features on the QTRAP® 5500 System provides the highest level of selectivity. The analyte ion of interest is isolated in the Q1 quadrupole with a user–selected resolution, usually unit resolution (0.7 Th FWHH). It is then fragmented in the Q2 collision cell, providing a broad range of product ions to be selected in the ion trap.
The in-trap fragmentation is achieved through the application
of a single wavelength / narrow band excitation. As shown in Figure 3, this allows very selective fragmentation. The C12 isotope of a product ion can be speci cally excited and fragmented to completion with minimal fragmentation of the C13 isotope.
This provides further selectivity advantages in the removal of interfering background. The combination of these features
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ORTHOGONAL SELECTIVITY TOOLS


provides unprecedented selectivity and exibility in the design of
3
the optimal MRM quanti cation experiments.
Sensitivity
Linear AcceleratorM Trap Technology has resulted in ground breaking improvement in the handling of ions inside the linear ion trap of the QTRAP® 5500 System, resulting in up to 100x more sensitivity. Trapped ions are manipulated within the linear ion trap through the use of auxiliary DC elds provided by the addition
of small electrodes (Figure 4, top). Ions are gently moved toward the extraction region of the linear ion trap during the cooling period by a voltage applied to the trap collar. A potential barrier
is created by increasing the potential on the auxiliary electrodes just before the mass scan to complete the ion concentration process. The application of this axial eld has a signi cant effect on sensitivity (Figure 4, bottom left).
In addition, a radio frequency is applied to the exit lens of the Linear Accelerator Trap resulting in further sensitivity gains (Figure 4, bottom right). These two innovations enable better than unit resolution to be obtained in the trap scan modes at these very high scan speeds.
Removal of Tough Interferences
Innovations in scanning speed, selectivity and sensitivity on the QTRAP® 5500 System enable successful implementation of the MRM3 work ow for a wide range of analytes 3,4. Sometimes, background noise or interferences can limit the detection of an analyte. Shown in Figure 5 is an example of an interference that has the same MRM transition as Clenbuterol and elutes at the
same retention time. Use of MRM3 can completely remove this interference and enable a much lower detection of this analyte.
Conclusions
MRM3 is an effective strategy for quantitation of analytes when high background or interferences make standard MRM quantitation dif cult (Figure 5).
MRM3 can be used to achieve similar LOQ‘s with less sample preparation and simpli ed or faster chromatography.
MRM3 has been successfully applied to the detection and quantitation of small molecules, peptides, and protein biomarkers.
MRM3 is signi cantly improved on the QTRAP® 5500 System technology making it a useful tool for quantitation in
tough matrices.
The QTRAP® 5500 LC/MS/MS System is the highest performance triple quadrupole and linear ion trap system available on the market today providing users with many powerful quantitative and qualitative tools.
References
1Collings BA, (2007) Fragmentation of ions in a low pressure linear ion trap. J. Am. Soc. Mass Spectrom. 18, 1459-1466.
2Collings BA, and Romaschin AR, (2009) MS/MS of ions in a low pressure linear ion trap using a pulsed gas. J. Am. Soc. Mass Spectrom. 20, 1714-1717.
3Fortin T. et al, (2009) Multiple Reaction Monitoring Cubed for Protein Quanti cation at the Low Nanogram/Milliliter Level in Nondepleted Human Serum. Anal. Chem. ePub. Oct 19, 2009.
4
Niessen J. et al, (2009) Human platelets express OATP2B1, an uptake transporter for atorvastatin.
Drug. Metab. Dispos. Fast Forward. Feb 23, 2009.
For Research Use Only. Not for use in diagnostic procedures.
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78
Improved Selectivity for the Low-Level
Quanti cation of the Therapeutic
Peptide Exenatide in Human Plasma
MRM3 quantitation for highest selectivity in complex mixtures on the AB SCIEX QTRAP®
5500 System
Yan Xu, John Paul Gutierrez, Tian-Sheng Lu, Haiqing Ding, Katie Piening, Erin Goodin, Xiuying Chen, Kristin Miller, Yong-Xi Li
Medpace Bioanalytical Laboratories, Cincinnati, OH
Key challenges of peptide quantitation
Insuf cient sensitivity – The best, previously reported LOQ is 100 pg/mL; extended-release pharmacokinetic studies demand lower levels of detection.
Limited quantitation range – Analytical range of ELISA-based method is <2 orders of magnitude; at least 3 orders of magnitude is desired in bioanalysis.
Low speci city – Complex biological matrices hamper data resolution and require sophisticated sample preparation and/or advanced instrumentation.
Systematic measurement errors – Especially for ultra-low level quantitation, measurement errors have a signi cant effect on data accuracy and precision.
Key bene ts of MRM3 peptide quantitation
High selectivity – Because of the multiple fragmentation steps in MRM3, the resulting spectra have lower backgrounds and fewer interfering, co-eluting contaminants.
Improved sensitivity – Detection limits in very complex matrices can often be improved using MRM3 analysis by removing interferences at the low end of the concentration curve.
Key features of QTRAP® 5500 System for MRM3 peptide quantitation
Fast scanning speed – Improvements to the QTRAP® 5500 Systems has enabled faster and more sensitivity MRM3 analysis.
Unique hybrid linear ion trap MS – Q1 is used for precursor ion selection (unit resolution), and Q2 for the rst fragmentation step in transmission mode. No low mass cut-off associated with the rst fragmentation step and higher collision energies delivers higher speed, greater selectivity, and more exibility in the choice of the rst product ion.
Dr. Yong-Xi Li
Figure 1: Structure of Exenatide. Exenatide is a large, 39- amino acid peptide
(MW = 4,186.6 Da) that acts as a regulator of glucose metabolism and insulin secretion.
Introduction
Pharmaceutical research is increasingly focused on the development of biotherapeutics, which in turn is driving increased interest in the use of LC/MS/MS techniques for the quantitative analysis of proteins and peptides. To measure these peptide therapeutics in plasma, researchers have traditionally relied upon ligand-binding-based assays such as ELISA for pharmacokinetic and drug development studies. However, these immunoassays
are fraught with selectivity problems, often displaying high background and a dynamic range too limited for accurate and precise concentration measurements at low levels. On the other hand, LC/MS/MS methods offer an increased dynamic range and fast method development, as well as signi cant improvements in the accuracy of bioanalysis.
Exenatide is a large therapeutic peptide that has been approved for the treatment of diabetes mellitus type 1 and 2. This peptide enhances glucose-dependent insulin secretion by the pancreatic beta-cell, acting as a regulator of glucose metabolism and
insulin secretion. In recent years, immunoenzymatic assays have been the primary method for quantitating exenatide, but the time-consuming development of antibodies, and the lack of precision and accuracy in quantitative measurements have compelled the development of new bioanalytical strategies for detecting peptides in biological uids. Because of its highly
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Figure 2: Schematic of MRM3 for quantitative analysis by LC/MS. The parent ion is rst selected in Q1 and then fragmented in Q2. Product ions are trapped and isolated in the linear ion trap, followed by resonance excitation for secondary fragmentation. Second-generation product ions are then scanned out to the detector.
selective capacity, an MRM3 LC/MS strategy1 was evaluated using the AB SCIEX QTRAP® 5500 System for reproducible and robust peptide detection in human plasma. We found that MRM3 scans (Figure 2) on a hybrid triple quadrupole linear ion trap mass spectrometer using high ow chromatographic conditions can provide high-throughput, as well as highly sensitive and selective, measurement of exenatide in biological uids, demonstrating the precision and accuracy necessary for the regulated laboratory environment.
Materials and methods
Sample preparation
Human plasma samples containing exenatide were dried using a gas vortexer (TurboVap) under nitrogen and reconstituted. In all
For Research Use Only. Not for use in diagnostic procedures.
steps, pH values and organic
Chromatography
LC system: Column:
Injection: Flow rate: Mobile phase:
Gradient:
Mass spectrometry
Mass spectrometry system:
phase were carefully controlled.
Shimadzu UFLC LC-20ACXR
Reverse phase C-18 (2.0 x 30 mm, 5 μm)
5 μL
0.6 mL/min
A) water, 0.1% formic acid
B) methanol, 0.1% formic acid
Time/min A% B% 0 98 2 0.5 98 2
5 5 95
AB SCIEX QTRAP® 5500 System
Figure 3: MRM3 assay design. An enhanced mass spectrometry (EMS) scan (top) is used to select the dominant parent ion; the most intense fragment ions are identi ed using enhanced product ion (EPI) mode (middle); and MS3 fragmentation is used to select the best secondary fragments to extract (bottom).
Figure 4: MRM3 signi cantly improved selectivity for exenatide in plasma over MRM analysis. Extracted ion chromatograms show the results of an MRM (top) and an MRM3 (bottom) scan for the detection for exenatide. Elimination of chromatographic interferences and background noise improves the LOQ for exenatide in plasma.
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LC/MS analysis using the MRM3 acquisition strategy.1 Using the MS3 scan, the trap was lled using dynamic ll time (DFT), and the instrument was scanned at 10,000 Da/sec. The trap excitation time was 25 msec, giving a total cycle time of 0.17 sec. MRM3 analysis used the transitions: 838→396→202
for exenatide quantitation.
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80
LLOQ
LQC
MQC
MHQC
HQC
Conc (ng/mL)
5.00 15.0 50.0 800 1800
5.14 15.9 46.4 779 1650
4.32 16.9 47.2 767 1549
5.65 12.0 41.7 821 1521
4.54 13.5 43.7 729 1641
3.69 17.1 50.0 658 1745
4.22 17.4 45.3 751 1672
Mean 4.59 15.5 45.7 751 1630
SD 0.701 2.22 2.85 54.8 82.4
%CV 15.3% 14.3% 6.2% 7.3% 5.1%
RE -8.2% 3.2% -8.6% -6.1% -9.5%
Figure 5: Calibration curves for exenatide quantitation in human plasma using MRM and MRM3 analysis. (Top) MRM measurements of exenatide (250-1,000 ng/mL) in plasma and (bottom) MRM3 measurements from 5–1000 ng/mL are compared. The graph displaying the MRM3 data shows signi cantly improved linearity (R2 = 0.996) over the data obtained by MRM analysis.
Table 1: Accuracy and precision of QC samples for exenatide quantitation. Data processing
Samples were acquired with the Analyst® 1.5 Software. Quanti cation was completed with MultiQuantTM Software, version 2.
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Results and discussion
MRM3 exenatide assay development
To determine the MRM3 transitions for exenatide quantitation, enhanced MS (EMS) mode was used to scan the plasma samples. The multiply-charged parent ion [M+5H]5+ at m/z 838.3 was selected as the rst precursor (Figure 3, top). Upon fragmentation, the resulting predominant product ion m/z 396.4 was selected as the second precursor ion (enhanced product ion (EPI) scan, Figure 3, middle) and then fragmented in linear ion trap to generate the MS3 spectrum (Figure 3, bottom). The major fragment ion m/z 202.2 was selected as the second-generation product ion for
MS3 quanti cation.
Assay performance for exenatide
Use of MRM3 analysis resulted in signi cantly improved selectivity for exenatide in human plasma extracts. Figure 4 shows a comparison of MRM3 vs. traditional MRM quantitation. Baseline was lower and chromatographic interference from the plasma matrix was completely eliminated in the MRM3 scan. The fast scanning speed of the QTRAP® 5500 System (10,000 Da/sec) provided a suf cient number of data points across the analyte’s chromatographic peak for good reproducibility. The improved detection performance resulted in excellent assay performance at the LLOQ and four QC levels as shown in Table 1. Accuracy and %CV for six replicates demonstrate that the MRM3 approach is capable of quantitative performance suitable for development- grade bioanalytical assays.
Conclusions
A sensitive and selective bioanalytical assay for exenatide in human plasma was successfully developed using MRM3 analysis on the QTRAP® 5500 System.
The increased selectivity of MRM3 eliminated baseline noise and chromatographic interferences, delivering analytical performance that was superior to traditional MRM scans.
MRM3 assays demonstrated the potential for excellent linearity achieving a dynamic range of 5–2,000 ng/mL, compared with a range of less than 250–1,000 ng/mL for traditional MRM.
Accuracy and reproducibility of the MRM3 assay met the requirements for a development stage bioanalytical assay.
Acknowledgements
The authors would like to acknowledge Gary Impey, Xavier Misonne, Johnny Cardenas, Suma Ramagiri, and Mauro Aiello from AB SCIEX for their support and valuable advice during the completion of this work.
References
1 MRM3 Quantitation for Highest Selectivity in Complex Matrices. AB SCIEX Technical Note, Publication 0920210-01
For Research Use Only. Not for use in diagnostic procedures.
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82
Quanti cation of Prostate Speci c Antigen (PSA) in Nondepleted Human Serum using
MRM3 Analysis
High-throughput analysis of protein biomarkers using the AB SCIEX QTRAP® 5500 System
Yan Fortin T1, Salvador A2, Charrier JP1, Lenz C3, Bettsworth F1, Lacoux X1, Choquet-Kastylevsky G1, Lemoine J2 1BioMerieux, France, 2University of Lyon, France, 3AB SCIEX, Germany
Introduction
Over the last ten years, protein biomarkers of disease were discovered – from a comparatively small number of proteomics samples – using mass-spectrometry-based applications. Veri cation and validation of these biomarkers has been
minimal, limiting the translation of these proteins into viable, routine clinical assays. Traditionally, clinical researchers have used immunoassays to quantify low-level proteins in biological samples; however, development of these tests is very time-consuming,
and the results often lack reproducibility and speci city. Analytical techniques that sustain the rapid development of proteomics assays with high speci city and sensitivity are needed, driving
a rapidly growing interest in LC/MS-based strategies for
protein quantitation.
The use of multiple reaction monitoring (MRM) scans combined with stable-isotope-labeled proteins and peptides for the quanti cation of proteins has been actively explored over the last few years and shows great promise for clinical applications. Some key requirements that must be met for MRM scans to
be applicable to the clinical environment are: high speci city, robustness, very high throughput, and high sensitivity – detecting low-abundance proteins in the ng/ml to pg/ml concentration range in human plasma. In keeping with the high-throughput laboratory environment, sample preparation must also be simple and robust.
Another limitation hindering the widespread use of MRM
for biomarker veri cation has been the use of nano ow chromatography. While it greatly increases the overall sensitivity of LC/MS/MS experiments and requires signi cantly reduced amounts of sample, nano ow chromatography does not offer the sample throughput, reproducibility, and robustness required for implementation in clinical research laboratories.
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Figure 1: MRM3 scans for quantitative analysis by LC/MS. Analyte precursor ions are selected in Q1 and fragmented in Q2; the resulting product ions are collected in the linear ion trap (LIT). A suitable fragment ion is isolated and fragmented in a second step using resonance excitation. Second generation product ions are collected and scanned out of the LIT to the detector.
To overcome these challenges, we developed a novel MRM approach that combines the use of fast- ow analytical chromatography with the new, highly-selective mass spectrometry technique, MRM3 – a scan mode that signi cantly reduces MRM background noise and spectral complexity by producing and analyzing second-generation product ions. This approach enables robust detection of low-level protein biomarkers from human serum at low ng/mL concentrations and provides an ef cient method of protein quantitation applicable for high-throughput, multi-sample analysis.
TORTHOGONAL SELECTIVITY TOOLS


Materials and methods
Sample preparation
After denaturation with 6 M urea and reduction with 30 mM dithiothreitol, human serum samples were alkylated with 50 mM iodoacetamide. Proteins were digested with trypsin overnight at 37 °C (1:30 w/w enzyme to substrate ratio). After desalting using reversed-phase cartridges (Oasis HLB, 3 cm3, Waters), samples were fractionated using an MCX cartridge with elution at pH 5.5 using a methanol/acetate-buffer mixture (Waters).2
For Research Use Only. Not for use in diagnostic procedures.
Chromatography
Column:
Flow rate: Mobile phase:
Gradient:
Mass spectrometry
Waters Symmetry C18 reversed phase (2.1 x 4100 mm, 3.5 μm)
300 μL/min
A) water, 0.1% formic acid
B) acetonitrile, 0.1% formic acid
Time/min A% B% 0 95 5 0.5 95 5
30 60 40
System:
Interface:
MRM and MRM3 scan settings
AB SCIEX QTRAP® 5500 System Turbo VM Source
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Figure 2: MRM3 assay design. The dominant parent ion is selected from an EMS scan (top), and the most intense fragment ions are identi ed using the EPI mode (middle), and MS3 fragmentation is used to select the best second-generation fragments to extract (bottom). Multiple second-generation fragments can be used to generate MRM3 XICs (lower panel).
Figure 3: MRM quanti cation of PSA (5 ng/ml) in human serum. High- ow HPLC coupled with MRM analysis was used to detect PSA after depletion, digestion, and fractionation of the serum sample.
MRM analysis was performed using unit resolution for both Q1 and Q3 quadrupoles. MRM3 analysis was performed using the MS/MS/MS scan function. The precursor ion was isolated in
Q1 using unit resolution. First generation product ions were generated in the Q2 collision cell using an optimized collision energy and trapped in the Q3 linear ion trap for 200 msec.
A suitable rst-generation product ion was subjected to resonance excitation for 25 msec to produce second-generation fragments. Ions were scanned out of the ion trap at 10,000 Da/sec (total cycle time 350 msec/peptide).2 Q0 trapping further increased sensitivity.
Data processing
Data was processed using MultiQuantM Software. MRM peak areas were integrated, either individually or summed together. MRM3 peak areas were determined by summing the integration of up to four granddaughter ions.
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Figure 4: Reduction in background interference with MRM3 analysis. PSA spiked into human serum (female) at 50 ng/ml was analyzed by MRM scan (left) or by MRM3 scan (right). A signi cant reduction in background was observed when an MRM3 scan was used for PSA detection.
Results and discussion
MRM detection of PSA
Recently, PSA in human serum was detected using MRM analysis on a 4000 QTRAP® System with high- ow chromatography.1 HSA depletion followed by mixed-cation-exchange fractionation and HPLC separation enabled low-level detection at 5 ng/mL (Figure 2). Depletion of highly-abundant proteins is dif cult to automate, and additional strategies were explored that removed this step and accelerated the work ow. The highly-selective MRM3 scan mode (Figure 2) was evaluated for its ability to maintain a high- level of sensitivity while enhancing analyte separation from co- eluting interferences. Detection of PSA and other low-abundance proteins in human serum at very low levels was facilitated by suf cient production of second-generation product ions in the linear ion trap of the QTRAP 5500 System; multiple product ions can be summed to generate MRM3 quantitative results, boosting the sensitivity of the assay (Figure 2, lower panel).
Using MRM3 scans for PSA quanti cation
To assess the intrinsic gain in speci city of the MRM3 method compared to the conventional MRM operating mode, a tryptic digest of human serum from a female donor was spiked with tryptic digests of bacterial proteins TP171, TP574, TP435, and core NS4, and the human prostate-speci c antigen (PSA) protein over a range of concentrations (0–1,000 ng/ml). One average,
the limits of detection for the ve model proteins improved by
3- to 5-fold when detected using the MRM3 scan mode over the traditional MRM mode.2 MRM3 analysis of the model proteins resulted in spectra with decreased background noise and fewer co-eluting interferences compared to spectra obtained by the regular MRM mode; representative spectra for PSA detected by both scan methods are shown in Figure 4, and clearly illustrate the heightened speci city for the analyte.
Figure 5: Improved detection of bacterial core NS4 peptide, ALESFWAK, in human plasma. MRM analysis yielded a quantitation limit (LLOQ) of 50 ng/ml (right panels). MRM3 detection resulted in an improved LLOQ of 10 ng/ml.
The selectivity gains from MRM3 scans positively impacted the quantitative data obtained from standard concentration curves constructed from MRM3 peak area data, which showed excellent linearity over three orders of magnitude for the core NS4 peptide ALESFWAK (Figure 5). The accuracy and precision of the MRM3 data for the core NS4 peptide met bioanalytical standards, giving a %CV between 0.8–16% for MRM3 data,an improvement over the %CV range of 0.4-19.3% for MRM data alone (Table 1).
An improved LLOQ was shown for the core NS4 peptide when using the more selective MRM3 experiment over the standard MRM – lowering the LLOQ to 10 ng/mL and enhancing the capacity of LC/MS/MS for very-low level peptide detection.
Concentration (ng/mL)
10
50
100
500
1000
MRM3 Mean 11.3 47.0 92.4 516.7 992.7
Precision 16.0 6.7 6.4 3.2 0.8
Accuracy 13.2 -5.9 -7.6 3.3 -0.7
MRM Mean 54.7 101.1 511.3 994
Precision 8.17 19.3 1.3 0.4
Accuracy 9.4 1.1 2.3 -0.6
Table 1: Accuracy and precision analysis of quanti cation data for the core NSA peptide, ALESFWAK, in human plasma.
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Conclusions
Protein quantitation in human serum at low ng/ml concentrations was demonstrated using high- ow chromatography and MRM3 analysis.
Critical detection levels of 4-10 ng/ml of circulating PSA were accurately determined in human serum using a robust two-step sample preparation protocol combined with MRM3 analysis.
Improvements in speed and sensitivity to the QTRAP® 5500 System make MRM3 analysis a robust quantitative strategy for peptides in complex matrices when signi cant background interferences are present.
References
1 Fortin T, et al. (2009) Clinical quantitation of prostate-speci c antigen biomarker in the low nanogram/milliliter range by conventional bore liquid chromatography-tandem mass spectrometry (multiple reaction monitoring) coupling and correlation with ELISA tests. Mol. Cell; 11(8): 1006.
2 Fortin T, et al. (2009) Multiple reaction monitoring cubed for protein quantitation at the low nanogram/milliliter level in nondepleted human serum. Anal. Chem; 15: 9343.
3 MRM3 Quantitation for Highest Speci city in Complex Matrices. AB SCIEX Technical Note, Publication 0920210-0.
For Research Use Only. Not for use in diagnostic procedures.
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UGT Family of Enzymes: Quanti cation of Tryptic Peptides
Accelerating MRM3 work ows on the QTRAP® 6500 System for enhanced selectivity in
complex matrices like tryptic digests
Suma Ramagiri, Loren Olson, Gary Impey, Carmen Fernandez-Metzler AB SCIEX, 71 Four Valley Drive, Concord, ON, L4K 4V8 Canada PharmaCadence Analytical Services, LLC Hatfield, PA 19440
Key challenges of peptide quantitation
Insuf cient sensitivity – The best, previously reported LOQ is 100 pg/mL; extended-release pharmacokinetic studies demand lower levels of detection.
Limited quantitation range – Analytical range of ELISA-based method is <2 orders of magnitude; at least 3 orders of magnitude is desired in bioanalysis.
Low speci city – Complex biological matrices hamper data resolution and require sophisticated sample preparation and/or advanced instrumentation.
Systematic measurement errors – Especially for ultra-low level quantitation, measurement errors have a signi cant effect on data accuracy and precision.
Key bene ts of MRM3 peptide quantitation
High selectivity – Because of the multiple fragmentation steps in MRM3, the resulting spectra have lower backgrounds and fewer interfering, co-eluting contaminants.
Improved sensitivity – Detection limits in very complex matrices can often be improved using MRM3 analysis by removing interferences at the low end of the concentration curve.
Key features of QTRAP® 5500 System for MRM3 peptide quantitation
Introduction
In Part 1 of this series, we introduced the UGT (UDP-glucuronsyl- transferase) family of enzymes and presented a framework
for understanding why quantitating UGTs is important for the drug discovery and development process.1 In short, UGTs are responsible for glucuronic acid conjugation of xenobiotics, an important route of elimination for over 200 drugs – making
this a pathway of common focus during the development of pharmaceuticals. Quantitation of UGT enzyme expression and its absolute cellular level provides pharmaceutical scientists with essential information for further characterization of tissues and cell lines used for drug metabolism studies and for studies on inductive pathways that effect UGT enzyme expression.
Improvements to standard MRM approaches for peptide quantitation are limited to adjustments in ion pairs, manipulation of different CE pro les, and development of alternate sample preparation and chromatographic strategies to enhance the
signal on analytes of interest. Often these adjustments are very time-consuming and don’t provide signi cant improvement over the original methodology. QTRAP® Technology offers MRM3 capabilities (Figure 1), providing an additional selectivity option for those analytes with a generous spectrum of fragment ions.
The common problems experienced when quantitating low-level peptides in complex matrices are a lack of sensitivity and the presence of variable background interferences. This tech note describes fast and novel strategies available on the QTRAP® 6500 Series of instruments that provide up to 5-fold better LOQs than the standard MRM approach – with no sacri ce in throughput. The MRM3 technique conducted on the QTRAP® 6500 System quickly and easily provides better selectivity and sensitivity for peptide detection – ultimately improving the ef ciency of assay development and execution for the bioanalysis of UGTs.
TCarmen Fernandez
T® Fast scanning speed – Improvements to the QTRAP
5500 Systems has enabled faster and more sensitivity MRM3 analysis.
Unique hybrid linear ion trap MS – Q1 is used for precursor ion selection (unit resolution), and Q2 for the rst fragmentation step in transmission mode. No low mass cut-off associated with the rst fragmentation step.
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Materials and methods
Sample preparation
Standard tryptic digestion procedures were applied to 10 lots of human liver microsomes. Standard curves were made using digested rat liver microsomes and BD Supersome Human UGT products.
Data processing
MRM3 scans are fragmentation pathway speci c routes to the ion of interest (Figure 1). The following MRM3 scans were used to quantitate UGT tryptic peptides: 1) for DIVEVLSDR y7 ion, 523.3/817.4/589.3; 2) for YIPCDLDFK y6 ion, 585.3/637.3/303.2, 3) for TILDELIQR y6 ion, 558.8/773.4/515.3 for RT 4.96 min. and 558.8/773.4/515.3 for RT 5.33. MRM3 scans were processed using Analyst® Software (Figure 2). All quantitative results were processed using MultiQuantM Software.
Results and discussion
Elimination of tryptic peptide background interferences using the MRM3 work ow
Analysis of tissue samples from human subjects can be problematic due to high-levels of variable background noise and co-eluting, interfering contaminants – and is especially true for the detection and analysis of low-levels of tryptic peptides in tissue matrices. To address these selectivity issues, a quantitative MS3-style acquisition mode, MRM3, can help remove interfering signals quickly. These MRM3 assays can be developed in minutes, an improvement over the lengthy amount of time (often days)
Figure 1. Description of MRM3 Scans on the QTRAP® 6500 System: The precursor ion is selected in Q1, followed by fragmentation in Q2. The residual precursor ion and resulting fragments are captured in the Q3 linear ion trap (LIT) for a designated ll time. A speci c fragment ion is selected for further analysis, and upon isolation in Q3. this ion is fragmented to form second-generation fragment ions. These ions are rapidly scanned out of the linear ion trap (LIT) and serve as the analytical signal for the MRM3 experiment. By reducing background and eliminating interferences, this technique results in LOQs almost an order of magnitude lower than analogous MRM. Compatible with fast ow rates, MRM3 acquisition can be combined with higher throughput chromatography, resulting in greater ef ciencies.
Chromatography
LC system: Column:
Column temp.: Injection:
Flow rate: Mobile phase:
Gradient:
Eksigent ekspert microLC 200
Eksigent® ChromXPM 3C18-300-CL (1.0 x 50 mm, 3 μm, 300 Å)
40 °C
5 μL
50 μL/min μL
A) water, 0.1% formic acid
B) acetonitrile, 0.1% formic acid Time/min A%
0 100
0.5 100
5 75
6 20
7.5 20
0 100
8 100
10 100
B% 0
0 25 80 80 0
0 0
For Research Use Only. Not for use in diagnostic procedures.
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THERAPEUTIC PEPTIDE BIOANALYSIS
Mass spectrometry
System: QTRAP® 6500 System
Used in positive MRM mode
Figure 2: Analyst® Software MRM3 acquisition method. An example of an Analyst® Software acquisition method showing ve MRM3 experiments including 10 MRM scans with a duty cycle of only 600 msec all in the same analytical run.
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Figure 3: A) An MRM-based chromatogram of the DIVEVLSDR.y7 tryptic peptide shows a background interference peak at RT 2.97 min and also one at RT 4.31, near the parent peak RT 4.38. B) The MRM3 technique used on the same peptide in 2A completely eliminated the interfering peaks (at RT 4.31 and 4.38), improving parent peak integration for a better %CV. C) An MRM-based chromatogram of the YIPCDLDFK y6 tryptic peptide shows an interfering signal at RT 4.29 min (~ 40% less intense than the parent peak at 4.50 min). D) The MRM3 technique completely eliminated the interfering peak (RT 4.29 min), improving the parent peak integration for better %CV.
Figure 4: MRM3 work ows for multiple second-stage UGT fragment ions provide selectivity gains. A) TheTILDELIQR.y6 tryptic peptide chromatogram shows two peaks at RT 4.96 min and 5.34 min for the MRM transition 558.8/773.4. B) An MRM3 scan (m/z 200-900) shows multiple ions (m/z 515.3 and 755.4) unique to the chromatographic peak, RT 4.96 min. C) The MRM3 chromatogram shows only one peak at RT 4.96 min for the transition 558.8/773.4/515.3+755.4. D) An MRM3 scan (m/z 200-900) shows multiple ions (m/z 529.4 and 658.4) unique to the chromatographic peak, RT 5.33 min. E) An MRM3 chromatogram shows no isobaric interference with only one peak at RT 5.33 min for the transition 558.8/773.4/515.3+755.4.
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Figure 5: Summing multiple UGT ions for increased selectivity and sensitivity.
A) Multiple, second-generation daughter ions from an MRM3 chromatogram
of UGT 2B7 peptide were summed (blue trace), enabling a ~2-fold increase
in signal intensity (Pink trace: chromatogram of only one second-generation ion). B) Linear calibration curve showing MRM3 transition for peptide UGT 2B17 (524.8/862.5/523.4).
typically required for sample preparation or chromatography improvements. To improve detection and quantitation over MRM- style experiments, MRM3 scans include an additional level of ion selection and fragmentation that removes or signi cantly reduces co-eluting interferences and background peaks, allowing for easier peak integration and shortened run-times for improved ef ciency. MRM3 chromatograms of two UGT tryptic peptides, the y7 DIVEVLSDR and y6 YIPCDLDFK, reveal the potential of this technique to simplify background, improve S/N, and increase throughput with faster chromatography (Figure 3).
Traditional MRM experiments may also produce spectra with multiple second-stage fragment ions, which impedes the straightforward quantitation of the peptide of interest. An extra level of selectivity gained from MRM3 experiments eliminates
the isobaric interferences and yields cleaner spectra for a higher degree of separation for the peak of interest. The close elution
of two peaks (from second stage fragment ions) was observed in the MRM chromatogram of the UGT y6 tryptic peptide TILDELIQR (Figure 4). MRM3 experiments on each of the peak removed the corresponding isobaric interference and facilitated further isolation of the single peak of interest.
Faster MRM3 scans for simultaneous, multi-analyte analysis at UHPLC speed
The QTRAP® 6500 System enables MRM3 scans that are twice
as fast as those conducted on previous generations of QTRAP® Technology, enabling faster chromatography and more MRM3 experiments for multiple analytes in a single injection. Automated MRM3 method-building makes de ning parameters effortless, while also making the MRM3 work ow fast, reproducible,
and easy-to-use – increasing throughput and selectivity at the same time. Faster MRM3 scans are achievable on QTRAP® 6500 System due to improved ion processing and manipulation along the ion path, resulting in faster cycle times. Improvements to
the detector capacity with IonDriveM Technology provide an increased linear dynamic range, producing calibration curves with an extended concentration range. Faster linear ion trap scan speeds of up to 20,000 Da/sec enable MRM3 scans compatible with UHPLC-compatible cycle times. This allows for extracted
ion chromatograms (XICs) of second generation product ions to be integrated with a suf cient number of data points across the chromatographic peak, increasing the precision and accuracy of the quantitative peptide data.
The sensitivity and selectivity of the MRM3 peptide quantitation assay can be further enhanced by summing multiple second stage fragment ions for an increase in signal intensity. A UGT 2B7 tryptic peptide, analyzed by MRM3, showed a two-fold increase in peak area when two second-generation ions were summed versus only one ion (Figure 5A). Quantitation data obtained from summing multiple MRM3 fragment ions was used to produce the calibration curve for the UGT 2B7 peptide (Figure 5B), which showed excellent linearity over three orders of magnitude.
Conclusions
MRM3 quantitation strategies offer the added bene t of a second stage of fragmentation over traditional MRM assays, raising the selectivity and sensitivity of detection for low-level peptides in complex biological matrices, such as tryptic digests of cellular extracts.
MRM3 scans improve signal-to-noise signals for analytes of interest in the most complex matrices by eliminating or reducing background interferences.
The faster MRM3 scan on the QTRAP® 6500 System accelerates acquisition time, making it compatible with UHPLC runs, while still maintaining a suf cient number of points across chromatographic peaks for highly precise and accurate quantitation of low-level analytes.
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Signature Peptide MRM Optimization
Made Easy for Therapeutic Protein and
TTPeptide Quanti cation
Ian Moore and Suma Ramagiri AB SCIEX, Concord, ON
Key challenges in MRM optimization for protein and peptide quanti cation
Choosing a unique peptide – A peptide that is unique to the protein of interest within a given background and is also suf ciently sensitive and selective.
Choosing the best MRM for the peptide – Multiple charge states are possible for a given peptide which in combination with the many product ion possibilities leaves many MRMs to be screened.
Optimizing MS parameters – Manual tuning can be tedious, and optimizing via LC injections is time consuming, particularly when monitoring multiple MRMs per peptide and multiple peptides per protein.
Key bene ts of using DiscoveryQuantTM Software for signature peptide optimization
Increase productivity with reduced method development time – By using an automated infusion or ow-injection based tune with DiscoveryQuantTM Software instead of LC with step parameters.
Save time with automated work ow – Predicted transitions from the Skyline software are validated on a real digest sample quickly. DiscoveryQuantTM Software is used to automatically optimize DP, CE, CXP and EP.
Figure 1: Time saving advantages of peptide optimization using DiscoveryQuantTM Software.
TTTTTTTTTProtein or mAb
In silico Digest
Figure 2: Schematic overview of the DiscoveryQuantTM Software tuning and optimization work ow.
Unique features of DiscoveryQuantTM Software for signature peptide quanti cation
Two experiment work ow – The QuickTune and FineTune experiments allow you determine the balance between throughput and ultimate sensitivity.
Always have access to your results – All results from a DiscoveryQuantTM optimization experiment are stored in the DiscoveryQuantTM database for easy retrieval and review.
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LC-MRM Injection Optimization
LC-MRM Injection to Screen for Peptides and Product Ions
LC-MRM Injections with Step Parameters for Optimization of DP, CE, CXP
DiscoveryQuantTM Software
Infusion or Flow Injection Tuning,
2 Minutes per Peptide
QuickTune product ions unknown
FineTune product ions known
QuickTune identifies: Precursor ion
Up to 7 product ions
A corresponding CE for each product ion
FineTune performs:
MRM based fine tuning to maximize sensitivity of: DP, EP, CE, and CXP Confirms precursor ion
User review and validation
Database (all optimized compounds)
160 to 240 minutes
30 minutes
SOFTWARE TOOLS


Global database sharing – The data from multiple systems can optimization that provides maximum quantitative sensitivity. The MM
be shared enterprise wide through the global DiscoveryQuant database connectivity.
Automatic MRM method generation – Using data from the database and DiscoveryQuantM Analyze optimized MRM methods can be generated automatically.
Introduction
Protein based therapeutics are a rapidly expanding component
of many pharmaceutical companies’ drug portfolio. Monoclonal antibodies (mAb) used in the treatment of cancer are one class
of protein therapeutics that has achieved success. In addition to proteins, smaller therapeutic peptides have achieved approval
for a wide variety of indications in metabolic, cardiovascular and infectious diseases. In order to support this rapidly expanding new class of drug molecules, the rapid development of sensitive and selective bioanalytical methods are required.
Historically, protein and peptide quanti cation has been done using ligand binding assays (LBA) but LBAs suffer from inherent variability, lack of speci city, narrow dynamic range, and time consuming method development. As an alternative to LBAs LC/MS/MS methods are both sensitive and selective, have a wide dynamic range, and have been a staple in the quantitation of small molecule drugs. Bioanalytical methods for proteins and mAbs generally require digestion of the sample with a proteolytic enzyme like trypsin followed by direct analysis of one or more of the proteolytic peptides. Unlike bioanalytical method development for small molecules the product ions of a peptide analyte can be predicted using known ion types (a, b, c, x, y, z). An excellent starting point for the development of an LC/MS/MS method
for peptides is Skyline software (MacCoss Lab Software), which will provide a list of the possible product ions of a given peptide plus an estimate of the DP of the precursor ion and a CE for the product ions. The next step in the method development is to determine which proteolytic peptides are actually produced by the digestion reaction, and which product ions are actually formed in the collision cell and for a given peptide. Lastly, for the product ions formed the CE and CXP needs to be optimized to achieve maximum sensitivity. DiscoveryQuantM Software is the ideal tool to perform this optimization. DiscoveryQuantM Optimize Software allows for optimization of compound dependent parameters via ow injection or infusion and then populates a database with these parameters.
DiscoveryQuantM Optimize Software offers two options for tuning and optimization: QuickTune and FineTune. The QuickTune experiment is used to identify product ions and is comprised of
a precursor ion scan, a DP optimization for the precursor and product ion scans at user de ned CEs. The product ion masses and an associated CE are then stored in the DiscoveryQuantM database. The FineTune experiment can then optimize DP,
CE, CXP and EP (Figure 2) using MRM transitions loaded from
the DiscoveryQuantM database for a seamless and automated
DiscoveryQuant database can also be manually populated with MRM information loaded from an external source like Skyline. In this way FineTune can be used to optimize DP, CE, CXP and EP without running a QuickTune experiment rst.
This technical note describes the results of experiments where DiscoveryQuantM Software was used to optimize compound dependent parameters and improve upon the sensitivity of methods obtained from the output of Skyline software for the quantitation of peptides.
Materials and methods
Sample preparation
Trypsin digested E. coli BGAL from the AB SCIEX mass spectrometer standards kit, Part No. 4368624 was diluted to
0.5 pmol/μL in 50% acetonitrile in water with 0.1%formic acid. Infusion was performed at 2 μL/min using an Eksigent microLC electrode (25 μm) in the Turbo VM ion source. LC/MS/MS injections were performed on a 0.10 pmol/μL (5 μL injection) sample at 0.25 mL/min with the standard AB SCIEX Turbo VM electrode.
Data work ow
The BGAL peptide sequence (UniProt #P00722 ) was pasted
into Skyline and in-silico digested with trypsin. Tryptic peptides between 9 and 25 amino acids were selected while excluding cysteine containing sequences. Skyline was setup to export up to six ‘y’ ions with masses above the doubly charged parent m/z. The molecular weight range of the peptides ranged from 1098.55 to 2445.97 Da. Only doubly charge peptides were selected and a list of doubly charged peptides and their ‘y’ ions was exported as an Analyst method in the .csv format. Using Excel, this .csv le was formatted into a table that could be imported to the DQ database and saved as a .txt le.
For Research Use Only. Not for use in diagnostic procedures.
LC conditions
LC System: Analytical column:
Analytical ow : Mobile Phase A: Mobile Phase B:
Shimadzu LC-30 Nexera System
Phenomenex Aeris Peptide XB-C18, 3.6 μ, 2.1 mm x 150 mm
0.25 ml/min
Water (0.1 % formic acid) Acetonitrile (0.1 % formic acid)
Gradient:
Time (min) 1.0 10.0 11.0 13.0 14.0
Mobile phase A% 97
50
5
5
97
Mobile phase B% 3
50
95
95
3
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Peptide
Monoisotopic Mass
Charge State
m/z
IDPNAWVER 1098.546 +2 550.3
TDRPSQQLR 1099.573 +2 550.8
HQQQFFQFR 1264.610 +2 626.8
ELNYGPHQWR 1298.616 +2 633.3
VDEDQPFPAVPK 1340.661 +2 650.3
LWSAEIPNLYR 1360.714 +2 671.3
LPSEFDLSAFLR 1393.724 +2 681.4
APLDNDIGVSEATR 1456.716 +2 697.9
QSGFLSQMWIGDK 1495.713 +2 729.4
YSQQQLMETSHR 1506.689 +2 748.9
LSGQTIEVTSEYLFR 1741.889 +2 754.4
VNWLGLGPQENYPDR 1756.853 +2 872.0
IENGLLLLNGKPLLIR 1775.103 +2 879.4
WSDGSYLEDQDMWR 1786.726 +2 888.6
LQGGFVWDWVDQSLIK 1889.968 +2 894.4
DVSLLHKPTTQISDFHVATR 2264.191 +2 946.0
YGLYVVDEANIETHGMVPMNR 2407.130 +2 1133.1
YDENGNPWSAYGGDFGDTPNDR 2445.973 +2 1204.6
Figure 3: DP and CE optimization data from the peptide APLDNDIGVSEATR using DiscoveryQuantTM FineTune. The CXP ramping data is shown below in Figure 4.
Figure 4: DP and CXP optimization data from the peptide APLDNDIGVSEATR using DiscoveryQuantTM FineTune.
Figure 5: Partial calibration curves of peptide APLDNDIGVSEATR. Maroon squares represent data from the DiscoveryQuantTM Software optimized LC-MRM method and blue diamonds represent data from the Skyline MRM method.
Table 1: List of the peptide sequences chosen for optimization. MS conditions
MS system: Ionization mode:
Software
Data acquisition: Quantitation:
QTRAP® 4500 System with a Turbo VTM Ion Spray Source
ESI with Positive Mode
DiscoveryQuantTM 2.1.2 Analyst® 1.6.1 Software MultiQuantTM Software
TTTResults and discussion
E. Coli BGAL (1024 amino acids) was digested in-silico with trypsin using Skyline and the tryptic peptides selected for optimization are shown in Table 1.
Skyline software assigned DPs in the range of 71 to 120 V for the precursor ions of Table 1 and CEs in the range of 27 to 66V for the 6 product ‘y’ ions of each precursor. These values were used to construct an LC-MRM method that was used to analyze a sample of the BGAL tryptic digest (0.10 pmol/μL). The LC peak areas for each peptide MRM were calculated and used for comparison.
The Skyline information (product ion masses, DP and CE) was imported into the DiscoveryQuantTM Software database. A FineTune experiment was then used to optimize: the DP between 5 and 150 V, the CE between ±20V of the Skyline assigned CE and the CXP between 2 to 30V. EP was not optimized and was kept at 10 V. Total infusion time for each peptide was 1.0 minute. At a ow rate of 2.0 μL/min ~40 μL of sample was consumed
or approximately 2.3 μg of protein. An example of the Optimize FineTune data (DP, CE) for peptide APLDNDIGVSEATR is shown in Figure 3. The Skyline CE for the product ions of this peptide was
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For Research Use Only. Not for use in diagnostic procedures.
Peptide
Q1/Q3
Retention Time (min)
0.10 pmol/μL BGAL
Avg. Area Gain (N=3)
Avg. S/N Gain (N=3)
DVSLLHKPTTQISDFHVATR
1133.1 /
1472.7 4.78 308% 296%
YGLYVVDEANIETHGMVPMNR
1204.6 /
1713.8 6.88 164% 157%
TDRPSQQLR
550.8 /
728.4 6.68 153% 147%
IENGLLLLNGKPLLIR
888.6 /
1023.7 6.89 91% 86%
APLDNDIGVSEATR
729.4 /
832.5 7.02 53% 55%
IDPNAWVER
550.3 /
660.3 6.04 49% 46%
YDENGNPWSAYGGDFGDTPNDR
1224.0 /
1754.7 3.81 48% 49%
YSQQQLMETSHR
754.4 /
760.3 7.37 31% 33%
LQGGFVWDWVDQSLIK
946.0 /
1289.7 7.15 31% 34%
ELNYGPHQWR
650.3 /
780.4 7.28 16% 13%
LPSEFDLSAFLR
697.9 /
1184.6 8.61 10% 11%
VDEDQPFPAVPK
671.3 /
755.4 8.20 9% 11%
WSDGSYLEDQDMWR
894.4 /
979.4 7.37 4% 4%
QSGFLSQMWIGDK
748.9 /
964.5 6.08 1% 0%
VNWLGLGPQENYPDR
879.4 /
1075.5 5.99 0% 2%
HQQQFFQFR
633.3 /
1000.5 5.30 -1% 1%
LWSAEIPNLYR
681.4 /
1062.6 5.98 -3% -4%
LSGQTIEVTSEYLFR
872.0 /
1143.6 5.62 -10% -10%
Figure 6: QuickTune results and product ion spectrum for peptide QSGFLSQMWIGDK.
Peptide
Q1/Q3
Retention Time (min)
0.10 pmol/μL BGAL
Avg. Peak Area (N=3)
Avg. S/N (N=3)
QSGFLSQMWIGDK 748.9 / 7.37 4.30E+04 3.65E+02 964.5
QSGFLSQMWIGDK 748.9 / 7.37 8.59E+04 7.48E+02 740.1
Gain 2.00 2.05
Table 3: Gains in peak area and signal to noise ratio for peptide QSGFLSQMWIGDK based on a product ion pair identi ed using QuickTune.
pair was selected from the database. The 0.10 pmol/μL sample was analyzed with this method and peak areas were compared to peak areas generated from the Skyline MRM method. The data in Table 2 shows the changes in peak area and signal to noise from the LC-MRM method generated using DiscoveryQuantM FineTune compared to the Skyline MRM method.
Of the 18 peptides tested 11 showed an increase in signal to noise ratio and peak area >10% and 7 were unchanged (±10%). The average gain for the 10 peptides showing improvement
was 94%. Calibration standards of peptides IDPNAWVER
and APLDNDIGVSEATR were prepared (Figure 5) and a partial calibration curve constructed over 3 orders of magnitude. Both the signal to noise and peak area gains was consistent across
all standards.
The QuickTune feature in DiscoveryQuantM Software includes
a product ion scan. Since not all peptide product ions can be described by a, b, c or x, y, z ion types the feature was used to analyze the BGAL digest for peptide product ions that are not supported in Skyline. In this work ow, only the peptide sequences need to be entered into the DiscoveryQuantM Software batch setup table. The QuickTune settings were set to scan for product ions from 700 amu up to the mass of the singly charged precursor ion using collision energies of 15, 25, 35, 45 and 55 V. In addition
Table 2: Changes in peak area and signal to noise ratio of peptides that were opti- mized with DiscoveryQuantM Software compared to un-optimized mass dependent parameters from Skyline.
35 V and the actual optimized CE was between 41 and 45 V for the 5 product ions.
DiscoveryQuantM Software ranks product ions in the database based on intensity of the CE ramping experiments. Therefore,
the most intense product ion is known prior to starting LC/MS/MS analysis. In the absence of library matching spectra this information is not known using Skyline alone. With the intensity of the product ions determined by the DiscoveryQuantM Software an LC/MS/MS method can be made for the digest sample including only the most intense MRMs for each peptide. This decreases the overall cycle time of the method and allows an increase in dwell time to improve S/N for each transition.
DiscoveryQuantM Analyze Software was used to build an LC-MRM method. Only the MRM of the most intense precursor/product ion
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to the product ion scans a DP ramp and an enhanced resolution precursor ion scan were performed. The data from peptide QSGFLSQMWIGDK are displayed in Figure 6.
The most intense product ion in the product ion mass window
is 740.1 amu. The product ion spectrum was visualized with PeakView® 2.0 software using Biotools and ion 740.1 was not assigned to either a, b, c or x, y, z ion types. The product ion
was included in an LC-MRM method and used to analyze the same 0.10 pmol/μL BGAL digest. The peak area and signal to noise ratio of the 748.9/740.1 pair was 2.02 fold greater that
the 748.9/964.5 pair. In addition to maximizing the sensitivity
of product ion types supported by Skyline, DiscoveryQuantTM Software can be used to increase the sensitivity of target peptides by identifying product ions not of the a, b, c or x, y, z type.
Conclusions
Optimizing the mass dependent parameters with DiscoveryQuantTM Software for transitions generated by Skyline increases the peak area and signal to noise ratio of the majority of peptides from a protein digest.
Optimizing peptides with DiscoveryQuantTM Software using infusion is fast, at 1 min per peptide, while requiring little sample, ~2 μL per peptide.
The QuickTune feature of DiscoveryQuantTM allows for the identi cation of product ions not of the traditional a, b, c or x, y, z ion types which can boost sensitivity for certain peptides.
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For Research Use Only. Not for use in diagnostic procedures.
© 2014 AB SCIEX. For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of AB SCIEX Pte. Ltd. or their respective owners.


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MultiQuantTM Software 3.0:
Peptide Bioanalysis for the Regulated
Bioanalytical Laboratory
Single software solution delivers an easy-to-use platform for peptide quantitation on diverse
MS instruments, while providing innovative tools for rapid data processing, improved data integrity and security, as well as unique audit trail functionality – for regulatory compliance
TKey challenges of software applications for peptide quantitation
Complicated software interfaces – Dif cult-to-navigate software screens make new user training challenging.
Non-integrated, multi-step work ows – Performing multiple peak quantitation functions using separate software programs is time-consuming and inef cient.
Tedious manual integration. Data integrity and authenticity.
Key bene ts of MultiQuantTM Software 3.0 for peptide quantitation
Increased ef ciency – MultiQuant Software can handle large data sets with thousands of MRM transitions for fast data processing and data review.
Fast, easy software training – Easy-to-use interface reduces the time required to train new operators for improved consistency.
Single software solution – Processes data from both small molecule drugs and biotherapeutics (proteins and peptides). Processes multiple data types (MRM, MRM3, TOF MS, SWATHTM Acquisition and MRMHR data) on multiple instruments (AB SCIEX Triple QuadTM Systems, QTRAP® Systems and TripleTOF® Systems).
Reduced manual data integration – Powerful and robust integration algorithms (MQ4) increase performance by automating peak integration with minimal manual intervention. Advanced query functionality quickly identi es samples that deviate from bioanalytical standards.
Improved data integrity and security – Ensures data integrity throughout with 21 CFR compliant features such as locking of results, secure reporting, robust Watson digital link, audit trail log and e-signatures. New and innovative audit trail functionality will allow easy search and present QA reviewers with track changes mode.
Key features of MultiQuantTM Software 3.0 for regulated bioanalysis
Easy to navigate screens – Fast, user-friendly interface with linked panes, custom queries, and one-click metric plots.
Compliance with regulatory audit tools – New audit trail with robust management and precise control.
Easy-to-access historical data – All versions of the calculated results are stored, and hyperlinked to the audit trail, allowing the reviewer to quickly see the data before and after a change.
Secure data reporting and exporting – Results table locking and secure export features ensure data integrity.
Searchable audit trail – Allows for a search of speci c events or samples across all results tables within a project for easier QA review.
Robust Watson digital link – Secure data transfer with audit trail log.
Introduction
Recent innovations in bioanalysis – such as automated sample preparation and ultra-high pressure liquid chromatography (UHPLC) – have positively impacted sample throughput for peptide quantitation experiments, accelerating sample processing as
well as data output. Peptide quantitation data analysis requires multiple, time-consuming, and labor-intensive steps including peak integration, review, and proper audit trail tracking. As a result, an upsurge in quantitation data can cause signi cant work ow bottlenecks, highlighting the need for rapid data processing and advanced automation of multi-step quantitative measurements and calculations to maintain a smoothly-running operation.
MultiQuantTM Software 3.0 quantitation package was designed for the bioanalytical laboratory with the goal of improving work ow ef ciency and facilitating regulatory compliance through an easy- to-use interface, powerful data analysis functions, and innovative audit/review features – cohesively integrated within Analyst® Software. MultiQuant Software handles large bioanalytical data
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Product features
Automation of quantitative functions
Automatic method creation – Peptides are routinely quantitated with high speci city using the multiple reaction monitoring (MRM) mode, and for complex samples, numerous MRM transitions
are monitored simultaneously in the same run. Setting up individual quantitation methods for multiple transitions is typically quite tedious and time-consuming, but MultiQuant Software streamlines method creation with a laborsaving process.
Using an Analyst Software ID column (Protein.Peptide.Fragment.IS Indicator) as a source for pre-de ned MRM transitions
(Figure 2), MultiQuant Software automatically creates individual MRM quantitation methods – eliminating the need to re-
enter peptide names or retention times. Peptide groups are automatically con gured, and peak elution times are de ned
by the retention times characterizing other MRM transitions for a particular peptide.
Automated absolute quantitation of peptides
For pharmaceutical research in the regulated environment, absolute quantitation of biotherapeutics is essential for regulatory compliance as well as for furthering pharmacokinetic research. Calculation of the absolute concentration requires information from a large number of samples, and as a result, can be quite monotonous and taxing, putting a strain on computing resources. To reduce the time-consuming aspects of absolute concentration determination, MultiQuant Software relies on pre-set functions
to generate calibration curves, to determine unknown sample concentrations, and to measure internal standards. Additionally, MultiQuant Software links calibration data sets obtained for endogenous peptides and their corresponding labelled standards for easier navigation amongst multiple standard curves for a particular peptide. Some of the speci c automated features for peptide standard curve generation include:
• Additional concentration and accuracy parameters for all the unknown samples calculated upon loading the calibration curve
• Straightforward generation of standard curves that are easily manipulated
• Creation of a statistics table for quick assessment of standard curve quality using %CV and accuracy (Figure 3, top).
• Instant creation of metric plots from results table data to visually assess data quality (Figure 3, bottom).
Intuitive software interface
M
Speed and ease-of-use – MultiQuant Software was designed
with an intuitive, easy-to-use interface for streamlined navigation of quantitation data – consolidating data functions for rapid processing and manipulation. The well-organized, user-friendly software format reduces the time required for new operator training, improving user consistency and con dence.
For Research Use Only. Not for use in diagnostic procedures.
sets using intuitive navigation panes, while retaining the identical software administration settings that were previously con gured in Analyst Software, such as access controls and user roles. The overlapping features between MultiQuant and Analyst Software greatly simplify many administrative functions, including software installation management, authorizations for access, data review privileges, and review of data uniformity, introducing a new level of time-savings into the regulated laboratory environment.
MultiQuant Software delivers a versatile and exible software solution designed for diverse analytes – from small molecules to larger biomolecules – with the capacity to process a wide variety of peptide quantitation data captured from MRM, MRMHR, and MRM3 scan functions, in addition to data from TOF MS and
full scan experiments. The intuitive software interface retains
an elegant simplicity, making software operation easy for any user regardless of experience level. Designed for multiple mass spectrometric platforms (including the QTRAP® Systems, Triple QuadM Systems, and TripleTOF® Systems), MultiQuant Software is incredibly adaptable, supporting even UV-DAD (ultraviolet diode array detector) and ADC (analog-to-digital converter) data sets, an application often overlooked in other conventional quantitation programs.
Here, we highlight the new innovations in this single software solution – and demonstrate how MultiQuant Software 3.0 signi cantly improves the speed and ease-of-use for quantitative peptide processing through the following functional upgrades:
• Additional automation of quantitation methods and calculations
• Intuitive software interface design for straight- forward navigation
• Fully-integrated and novel audit trail functionality for achieving regulatory compliance
• Enhanced validation procedures for fail-safe data integrity and secure reporting
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Figure 1: Easy-to-use interface. Intuitive user workspaces make data navigation easy, with dynamic linking of results panes, analytes, integration and one-click metric plots.
Figure 2: Automatic creation of quantitation methods. Using a naming convention for proteins and peptides pre-de ned in Analyst® Software, MultiQuant Software automatically builds quanti cation methods (bottom) based on the MRM method ID column (top). Multiple MRM transitions per peptide are considered part of a single analyte group for automatic retention time determination and interference analysis.
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The navigation panes in MultiQuant Software make it easy to transition between information screens for different analytes.
In addition, users can control display features with a high level
of speci city, presenting internal standard and analyte results alone or combined, along with selective review of speci c MRM transitions. Users can quickly build new data processing methods and evaluate the data in dynamically linked panes, automatically highlighting the chromatographic peaks and the corresponding integration when a sample is selected (Figure 4). In a single click, the user can view all analytes or a speci c analyte. The results tables and peak review are automatically updated.
Fully-integrated audit trail management
Innovative audit trail features – Accurate and secure record keeping is essential for the regulated laboratory, but the additional documentation steps required for an audit trail often put burdensome demands on resources, introducing additional, time- consuming procedures into the work ow. MultiQuant Software 3.0 presents a new, robust audit trail, with innovative features such as Grouped Audit Events, providing powerful audit trail management and ef cient review functionality integrated directly into the quantitation methodology. By consolidating required audit trail steps, signi cant time savings are realized, speeding up and automating the peptide quantitation process.
The new Audit Map Editor allows the laboratory administrator precise control over audit trail functionality using an intuitive, well-designed interface (Figure 5) to ensure consistency in the record-keeping process. For example, when a user is prompted
to enter a reason for a speci c auditable event, the administrator can pre-de ne the selections in a drop-down menu, limiting the user’s choices. The user can be allowed enter any reason without restrictions, or users can be constrained to a speci c list of allowed reasons. Furthermore, an administrator can ne tune the audit process by con guring reason selection options separately for each auditable event.
MultiQuant Software 3.0 introduces an additional level of
time savings and ef ciency by combining audit trail tasks and curbing the number of user prompts just to related events. For example, the number of electronic signatures collected during data processing is reduced through the Grouped Audit Events function (Figure 6), which merges the individual responses to selected auditable events into one all-encompassing e-signature. When the user enters the expected concentration values for a multi-point standard curve, the software will prompt only once for the electronic signature, instead of prompting at each point; however, the audit trail will still document each recognized event separately. Other Grouped Audit Events include multiple, discrete changes to the integration parameters, whereby each change is recorded individually in the audit trail, but the operator must only provide one signature, rather than multiple signatures, for every parameter update. This consolidation of tasks improves the speed of data processing without sacri cing compliance.
Figure 3: Peak area statistics table and metric plots. With a single click, useful tables of statistics (top) or informative metric plots (bottom) can be quickly generated for visual assessment of data quality. The change in area of the internal standard for one peptide is plotted versus injection number (bottom). Multiple peptides or multiple MRMs can be plotted by selecting multiple rows from the left hand navigation bar (top).
For Research Use Only. Not for use in diagnostic procedures.
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Figure 4: One stop easy data integration and regression set up. Key data processing parameters such as RT width, noise percentage, curve tting etc will be chosen up to process 1000’s of sample batch unattended.
Figure 5: New audit map editor. The editor provides intuitive control over auditable events. The administrator can specify which events are audited. For each event, a list of pre-de ned reasons can be speci ed and e-signatures can be required.
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