Research paperMALDI Immunoscreening (MiSCREEN): A method for selection of anti-peptide monoclonal antibodies for use in immunoproteomics
Graphical Abstract
Introduction
There is currently a shortage of antibodies for use in a variety of proteomics applications. The lack of such reagents presents a serious bottleneck for measuring the large number of different proteins in biological samples including tissues and plasma, the major source of biomarkers used in clinical diagnostics. Several ambitious projects are underway in both Europe (http://www.hupo.org/research/hai/; www.proteomebinders.org) and the USA (http://antibodies.cancer.gov) to make and characterize antibodies for use in immunohistochemical assays and for immuno-enrichment of proteins from complex mixtures. Such antibodies will be useful for expression analysis of proteins in tissues and cells and in “top-down” proteomics methods where intact protein targets are enriched before analysis by methods such as mass spectrometry, a method pioneered by Nelson et al. (1995).
Much less thought and effort has been focused so far on the development of anti-peptide antibodies suitable for quantitation of signature peptide surrogates of proteins from digests of complex biological materials. These kinds of antibody reagents are used in quantitative assays such as immuno-Matrix-Assisted Laser Desorption/Ionization (i-MALDI; Raska et al., 2003, Jiang et al., 2007) or Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA; Anderson et al., 2004). These applications use antibodies for specific enrichment of tryptic peptides from digests that are much more complex (in terms of number of different molecules present) than the protein mixtures from which they are derived. The unique requirements of such assays make it a challenge to derive and select antibodies with the desired characteristics. Essentially, such antibodies must be able to bind the target peptides from complex tryptic digests (typically from human plasma) and retain them through the washing steps prior to peptide elution and mass spectrometric analysis. Thus these antibodies must have high affinities, or more specifically, low off-rates for their peptide analytes. With current SISCAPA assays, the retention time required for effective peptide enrichment is a minimum of 10 min during which time unbound peptides are washed away. The MiSCREEN method described in this manuscript allows selection of such anti-peptide reagents.
A project to make quantitative assays for all human proteins, based on surrogate peptides, anti-peptide antibodies and mass spectrometry, has been proposed by Anderson et al. (2009). This human Proteome Detection and Quantitation Project (hPDQ) would function as an assay resource available worldwide. The project would require both MS-friendly proteotypic tryptic peptides for each target protein and renewable anti-peptide reagents specific for each. Currently, few of such anti-peptide reagents are available and the capacity to produce them is limited. Although some commercial and academic research labs claim to be able to make antibodies (or other affinity reagents) with high throughput, this has not been widely demonstrated for anti-peptide reagents with the performance characteristics required for use in immuno-MS assays.
We previously developed a surface plasmon resonance (SPR) method that allows selection of monoclonal anti-peptide antibodies that are able to bind tryptic peptides in solution phase and that are suitable for immuno-MS assays (Pope et al., 2009). This method measures true antibody affinities (not avidities) and is useful for kinetic analysis of small numbers of mAbs. However, the assay is too slow, cumbersome and expensive for high throughput screening.
We therefore sought to develop a method that would allow selection of high affinity anti-peptide mAbs (more specifically, those with low off-rates) in a more cost effective and high throughput fashion. To do this we have developed a method called MALDI immunoscreening (MiSCREEN) for rapid screening of hybridoma supernatants. The method allows the identification of antibodies that are able to bind specific peptides in solution phase from complex mixtures and that have low dissociation constants (kd) suitable for ultimate use in immuno-MS assays.
Section snippets
Peptides
Synthetic tryptic peptides chosen as surrogates of protein biomarkers were used throughout. Peptides that occur in a single protein encoded within the human genome and that yield several, strong multiple reaction monitoring (MRM) transitions in a triple quadrupole mass spectrometer were selected (Anderson et al., 2004). Peptides were synthesized by solid-phase methods by either the Chinese Peptide Company (Hangzhou, China) or by the UVic-Genome BC Proteomics Centre (Victoria, BC) and were
Synthetic peptides
It was important that all peptides synthesized for this work be of high-purity and that they be handled in a way that would preserve their integrity and prevent post synthesis modifications. Initially, peptide vendors were instructed that 80% purity was required and that both MALDI-TOF traces and HPLC traces be supplied. For almost all peptides, these criteria were met. However, further quality control was undertaken whereby after thawing and just before the use of the peptides, they were
Conclusions
Although anti-peptide mAbs are made worldwide, very few laboratories or companies have established methods to select those that bind short (e.g., tryptic) peptides with high affinity. Such antibodies are required for several different immuno-MS assays used for measurement of protein biomarkers in complex peptide mixtures. Often ELISA assays using peptides coupled to carrier proteins are used for anti-peptide antibody screening, although such assays measure avidity rather than true affinity
Acknowledgements
We thank Immunoprecise Antibodies Ltd and Jennifer Reid for the gifts of anti-peptide mouse mAbs and Leanne Ohlund for helping us with the Applied Biosystem-MDS SCIEX 4800 MALDI-TOF/TOF instrument. The authors acknowledge support from the US National Cancer Institute's Clinical Proteomic Technology Assessment for Cancer program (grant U24-CA126476-01) and from the Canadian Institutes for Health Research (grant MOP 81267 to TWP). The Kingfisher magnetic particle processor was supplied by
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Current Address: Institute of Molecular Systems Biology, ETH Zurich, HPT C 76 Wolfgang-Pauli-Strasse 16, 8093 Zürich, Switzerland.