Universität Tübingen
Plasma Proteome Analysis Using Peptide Group-Specific Immunoprecipitation "Triple X Proteomics"
Abstract
Blood plasma, the largest human proteome is readily accessible and can be minimalinvasively sampled making it a valuable source of potential biomarkers. However, its analysis is complicated by its complexity and the huge dynamic concentration range of its constituents. Automated assays were established using magnetic microspheres and customized group-specific triple X proteomics (TXP) antibodies. These antibodies are directed against short N- or C-terminal epitope motifs for the immunoprecipitation of signature peptides derived from groups of trypsin-digested plasma proteins. The introduction of these antibodies led to a very efficient enrichment of the targeted analytes and a reduction in sample complexity similar to the SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies) approach introduced by Anderson et al. (2004). The quantitative evaluation as targeted approach, via nano liquid chromatography multiple reaction monitoring and stable isotopes peptide standards revealed limits of detection of 21 ng/ml and limits of quantification of 189 ng/ml in a tryptic plasma digest assay. Two peptides with the same TXP tag were simultaneously captured with one TXP antibody in a multiplex dynamic range of about 7,000 (5 fmol peptide were still detectable in presence of 33.3 pmol matrix peptide). The qualitative evaluation as discovery approach, of a tryptic plasma digest assay via matrix-assisted laser desorption/ionization time of flight (MALDI TOF) analysis revealed the presence of TXP tag-specific peptides captured by the used TXP antibodies. Their analysis, however, was compromised by peptide peaks from non-targeted high abundant proteins. Sucrose gradient ultracentrifugation showed a 99% depletion of non-targeted peptides. Further investigations with a total of 14 TXP antibodies enabled the detection of targeted TXP peptides and a significant reduction in the intensity of non-specific peptides. TXP Proteomics by peptide group-specific immunoprecipitation coupled to mass spectrometry read-out allowed reproducible multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques. Furthermore, the application of TXP antibodies requires significantly fewer antibodies for immuno-affinity mass spectrometry based plasma proteome analysis compared to the use of anti-peptide antibodies.
Author and committee
dc:creator, dc:contributor.*- Author
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- Schneider, Sonja
Identifiers
dc:identifier.*- Identifier
- hdl:10900/49666