Accelerating ligand identification - Iowa State University

SCIENTIFIC REVIEW 2

Accelerating ligand identification

Reprinted from BIAjournal 5 (2) 1998

Lori Fitz, Stephanie Cook,
Elliott Nickbarg, Jack H. Wang
and Clive R. Wood.
Genetics Institute Inc.,
Cambridge, MA USA

Biacore-based protein-protein
interaction assays have been used
to search for growth factor
ligands of orphan receptors.
However, once a complex source
of a ligand, such as a cell line
conditioned medium, has been
identified, the rate determining
step is the purification of the
ligand for amino acid sequencing.
We have demonstrated that
Biacore can be used as a
'nanoscale' affinity purification
matrix using soluble receptors
immobilized on the sensor chip
leading to direct analysis by
tandem electrospray mass
spectrometry.

SCIENTIFIC REVIEW 2

Figure 1 Biacore technology has proven successful for shows typical results from a primary screen
the discovery of novel growth factors [1,2]. with four different R-Fc fusion constructs.
Cell line conditioned medium This work has typically involved These data show that interleukin-11 (IL-11)
screen for binding to 4 different immobilizing soluble forms of the extra- is readily detected in previously well
R-Fc: Kit-Fc, Flt4-Fc, IL-13Rα2-Fc cellular regions of orphan receptors to the characterized sources such as the human
and IL1 1Rα-Fc. The amount of carboxymethyl dextran surface of a sensor lung fibroblast, MRC5 [3] and the bladder
each R-Fc coupled to the chip chip and searching for their corresponding cell carcinoma, 5637 [4] when IL-11
surface is shown in the inset box. ligands by detecting changes in surface receptor a-chain-Fc (IL-11Ra-Fc) is
All cell line and peripheral blood plasmon resonance (SPR). These soluble immobilized on a sensor surface, and that
mononuclear cell (PBMC) forms of orphan receptors have typically IL-13 is detected in peripheral blood
conditioned media were been fusions of the extracellular region of mononuclear cell conditioned media using
concentrated up to 100-fold. each receptor linked to the Fc portion of the high affinity IL-13 receptor fusion
human immunoglobulin g1 (R-Fc). protein, IL-13Ra-Fc [5]. The binding
observed in this screen to Flt4-Fc led us to
In attempting to find ligands such as purify this ligand and isolate the corres-
cytokines present at low concentrations in ponding VEGF-C cDNA [1].
cell conditioned media, the sensitivity of this
assay is critically important. In our hands, Once a cell source of a ligand has been
conditions have been optimized to detect as identified, the conventional next step in
little as 2-10 ng/ml of ligand. Cell line identification of the ligand polypeptide
conditioned media for testing are entails the time and resource consuming
concentrated at least 100-fold, thus allowing process of producing large volumes of
the detection of ligands at starting conditioned media followed by receptor
concentrations as low as 20-100 pg/ml. This affinity chromatography and amino acid
is a sensitivity range that has allowed us to sequencing. In some cases, obtaining enough
detect successfully the ligands of a number starting material to purify the ligand to homo-
of different receptor tyrosine kinases and geneity in sufficient quantity is not possible,
hematopoietin receptors. Specificity of especially when ligands are discovered in
binding is analyzed by injecting cell line primary body fluids available in very limited
conditioned media over human IgG and quantities, such as synovial fluid.
other R-Fc coupled to Sensor Chip CM5,
and by competition with various co-injected If sufficient ligand can be recovered from the
R-Fc. surface of a Biacore sensor chip, then it may
be possible to identify ligands using mass
LIGAND FISHING WITH BIACORE 2000 spectrometry and circumvent the need for
purification scale-up. Previous work has
The configuration of Biacore 2000 allows successfully demonstrated that proteins
each sample to be analyzed in series over four bound to a sensor surface can be charact-
different R-Fc coupled surfaces. Figure 1 erized by matrix-assisted laser desorption/
ionization mass spectrometry (MALDI/MS)
Cell line conditioned media either with proteins bound to the chip
surface [6] or eluted from the surface [7].

Since most mass spectrometric techniques
can only develop complete sequence
information on small peptides, polypeptides
purified on a Biacore sensor surface would
need to be enzymatically digested in order to
be identified. In a tandem mass
spectrometer, such as a triple quadrupole
instrument, peptides can be sequenced by
selecting a peptide ion of interest in the first
mass analyzer, fragmenting in a collision
cell, and analyzing the resulting fragment
ions using the second mass analyzer. The
mass differences between the series of
fragment ions provide information about the

SCIENTIFIC REVIEW 2

identity and sequence of the amino acids in
the peptide. The mass selection capability
allows a tandem mass spectrometer to
analyze multiple peptides in a single sample.
In addition, a tandem mass spec-trometer
utilizing electrospray ionization can be
coupled to on-line purification techniques
such as reversed-phase high performance
liquid chromatography (RP-HPLC) or
capillary electrophoresis (Fig. 2).

AFFINITY PURIFICATION standards and selected for mass Figure 2
spectrometry. After digestion, using an Peptide Sequencing by using on-
WITH BIACORE 2000 automated in-gel tryptic digestion robot, line RPHPLC with triple quadrupole
peptide extracts were fractionated by tandem MS.
In order to develop a method to exploit the micro-capillary RP-HPLC using custom 75
Biacore sensor chip as an affinity mm diameter columns, and then were Figure 3
purification surface for subsequent tandem detected and sequenced automatically by a IL-13 binding on IL-13Rα1-Fc
electrospray mass spectrometry (ES/MS), we Finnigan TSQ-7000 triple quadrupole a. IL-13 binding on IL-13Rα1-Fc
have used high affinity IL-13Ra2 (KD = 0.5 mass spectrometer as they eluted from the (red) vs. IL-11Rα-Fc (blue)
- 1.2 nM) and lowaffinity IL-13Rα1 (KD = RP-HPLC column [9,10]. b. Silver stain SDS-PAGE of sensor
2-10 nM) binding chains of the murine IL- In order to identify known peptides, the chip eluates from the IL-13Rα1-Fc
13 receptor as a model system with a peptide fragmentation spectra generated and IL-11Rα-Fc (neg.)surface.
Biacore 2000 system [5,8]. The IL-13Rα-Fc by the mass spectrometer were compared
or a negative control R-Fc was immobilized against theoretical spectra calculated from b
at a high concentration (>10,000 RU per the protein sequence database OWL [11]
flow cell) on all the four flow cells of a using the program SEQUEST [12]. As shown
research grade sensor chip CM5 using amine in Figure 4, IL-13 was successfully identified.
coupling. Recombinant IL-13 was added to
100 fold concentrated cell conditioned a
medium to a final concentration of 10
µg/ml, injected over all four flow cells in
series for 10 minutes and then washed with
1 M sodium chloride for 30 seconds (Figure
3a). Bound protein was eluted with a pulse
of 0.1% (v/v) trifluoroacetic acid, pH 2 and
eluate was collected via the recovery cup of
Biacore 2000. This cycle was repeated 5
times and eluted material was recovered into
the same tube at the end of each cycle. As a
control, this procedure was repeated using
the IL-13 supplemented cell conditioned
medium but substituting the IL-13Rα1-Fc
surface with a surface conjugated to a
different hematopoietin R-Fc that does not
bind IL13: i.e. IL-11Rα-Fc. Approximately
500 RU was bound to the IL-13Ra1-Fc per
cycle while no binding was detected for IL-
11Rα-Fc.

Samples eluated from Biacore 2000 were
further purified by SDS-PAGE and visualized
by silver-staining (Fig. 3b). A polypeptide
species co-migrating with IL-13 standards
was found in the eluate from the IL-13Rα1-
Fc surface, but not in that from the IL-
11Rα-Fc surface. This specific band was
present at a nanogram level relative to IL-13

SCIENTIFIC REVIEW 2

Figure 4
Murine IL-13 identification. Peptide
ions from the electrospray mass
spectrometer were automatically
fragmented. The fragment data and
SEQUEST were used to screen the
OWL database for protein identity.

References AFFINITY PURIFICATION WITH proliferates in response to IL-13. The results
1. Fitz, L.J. et al. (1997) Oncogene obtained are shown in Figure 5. The eluate
15 613-618. BIACOREPROBE from the sensor probe was biologically
active to a level similar to a 10 ng/ml IL-13
2. Bruno J. (1997) BIAjournal We expected that the sensor surface of standard. Thus, this semi-quantitative
Special Issue 24-27. Biacore-B probe would also be useful for analysis revealed that the eluted IL-13 had
affinity purification since it has a larger retained most of its biological activity
3. Paul, S.R. et al. (1990) Proc. Natl. surface area for binding [13]. during the procedure.
Acad. Sci. USA 87 7512-7516.
We have used this system to purify IL-13 ACCELERATING LIGAND IDENTIFICATION
4. Lu, A.U. et al (1994) J.Immunol. from concentrated cell conditioned medium
Methods 173 19-26. containing 1µg/ml IL-13. High concentrations In conclusion, this model system
of IL-13Ra2-Fc were covalently coupled to demonstrates that soluble receptors
5. Donaldson, D.D. et al. (1998). J. the sensor probe surface, and similar binding, immobilized on the surface of a sensor chip
Immunol. 61 2317-2324. wash and elution conditions were used to or a sensor probe can be used as an affinity
those employed for Biacore 2000. Bound surface for purification of nanogram
6. Nelson, R.W. et al. (1997) Anal. material was eluted with 0.1% (v/v) quantities of growth factor ligands.
Chem. 69 4363-4368. trifluoroacetic acid, pH 2. Analysis by SDS- Furthermore, the elution and sample
PAGE and silver staining indicated that handling procedures used are satisfactory
7. Sonksen C.P. et al. (1998) Anal. approximately 10ng of IL-13 had been eluted for direct analysis by ES/MS. At least in the
Chem. 1998 70 2731-2736. with a very low background of contaminants case of IL-13, the functional activity of the
(data not shown). The eluate was assayed ligand is preserved during the procedure,
8. Hilton, D.J. et al. (1996) Proc. for its bioactivity using a cell line that permitting monitoring of the purification
Natl. Acad. Sci. USA 93 497-501. using biological assays. While both Biacore
2000 and Biacoreprobe have been used
9. Ashman et al. (1997) Lett. Pept. successfully, the larger surface area of the
Sci. 4 57. sensor probe and its simple, hand-held
operation minimizes the loss of starting
10. Davis, M.T. (1995) Anal. Chem. material. Therefore, in future work, Biacore
67 4549-4556. 2000 will be the system of choice for
identifying a suitable ligand source and
11. Bleasby, A.J. and Wootton, J.C. performing control experiments, while
(1990) Prot. Eng. 3 153-159. Biacoreprobe is preferred for affinity
purification and recovery of ligands for
12. Yates, J.R. 3rd (1998) ES/MS sequencing.
Electrophoresis 19 893-900.

13. Nelson, R.W. et al. (1997) Anal.
Chem. 69 4369-4374.

Figure 5

IL-13 recovered from Biacoreprobe
is bioactive. Dilutions of eluate
from an IL13Ra2-Fc Biacoreprobe
surface were compared to dilutions
of 10ng/ml IL-13 on transfected
Ba/F3.a1 cells responsive to this
cytokine. Proliferation was measured
after 3 days by 3 H-thymidine uptake.

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