Abstract
Targeted mass spectrometry using selected reaction monitoring (SRM) has emerged as the method of choice for the validation in blood serum, plasma, or other clinically relevant specimens of biomarker candidates arising from comparative proteomics or other discovery strategies. Here, we describe a method in which N-glycosites are selectively enriched from biological specimens by solid phase capture and PNGase F release, and then analyzed by SRM. Focusing the highly sensitive targeted mass spectrometry method on a subproteome enriched for secreted and shed proteins reproducibly identifies and quantifies such proteins in serum and plasma at the low nanogram per milliliter (ng/mL) concentration range. This protocol is intended to give an introduction to SRM-based targeted mass spectrometry with a special focus on the validation of biomarker candidates.
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References
Ahn, S.M., and Simpson, R.J. (2007) Body fluid proteomics: Prospects for biomarker discovery, Proteomics Clin. Appl. 1, 1004–1015.
Anderson, N. L., and Anderson, N. G. (2002) The human plasma proteome: history, character, and diagnostic prospects, Mol Cell Proteomics 1, 845–867.
Schiess, R., Wollscheid, B., and Aebersold, R. (2009) Targeted proteomic strategy for clinical biomarker discovery, Mol Oncol 3, 33–44.
White, C. N., Chan, D. W., and Zhang, Z. (2004) Bioinformatics strategies for proteomic profiling, Clin Biochem 37, 636–641.
Zhang, H., Li, X. J., Martin, D. B., and Aebersold, R. (2003) Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry, Nat Biotechnol 21, 660–666.
Zhang, H., Liu, A. Y., Loriaux, P. et al. (2007) Mass spectrometric detection of tissue proteins in plasma, Mol Cell Proteomics 6, 64–71.
Wollscheid, B., Bausch-Fluck, D., Henderson, C. et al. (2009) Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins, Nat Biotechnol 27, 378–386.
Stahl-Zeng, J., Lange, V., Ossola, R. et al. (2007) High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites, Mol Cell Proteomics 6, 1809–1817.
Lange, V., Picotti, P., Domon, B., and Aebersold, R. (2008) Selected reaction monitoring for quantitative proteomics: a tutorial, Mol Syst Biol 4, 222.
Mallick, P., Schirle, M., Chen, S. S., et al. (2007) Computational prediction of proteotypic peptides for quantitative proteomics, Nat Biotechnol 25, 125–131.
Kuster, B., Schirle, M., Mallick, P., and Aebersold, R. (2005) Scoring proteomes with proteotypic peptide probes, Nat Rev Mol Cell Biol 6, 577–583.
Addona, T. A., Abbatiello, S. E., Schilling, B. et al. (2009) Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma, Nat Biotechnol 27, 633–641.
Anderson, L., and Hunter, C. L. (2006) Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins, Mol Cell Proteomics 5, 573–588.
Kuzyk, M. A., Smith, D., Yang, J. et al. (2009) Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma, Mol Cell Proteomics 8, 1860–1877.
Picotti, P., Bodenmiller, B., Mueller, L. N. et al. (2009) Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics, Cell 138, 795–806.
Picotti, P., Lam, H., Campbell, D., Deutsch, E. W. et al. (2008) A database of mass spectrometric assays for the yeast proteome, Nat Methods 5, 913–914.
Picotti, P., Rinner, O., Stallmach, R. et al. (2010) High-throughput generation of selected reaction-monitoring assays for proteins and proteomes, Nat Methods 7, 43–46
Desiere, F., Deutsch, E. W., Nesvizhskii, A. I. et al. (2005) Integration with the human genome of peptide sequences obtained by high-throughput mass spectrometry, Genome Biol 6, R9.
Deutsch, E. W., Lam, H., and Aebersold, R. (2008) PeptideAtlas: a resource for target selection for emerging targeted proteomics workflows, EMBO Rep 9, 429–434.
Lam, H., Deutsch, E. W., Eddes, J. S. et al. (2007) Development and validation of a spectral library searching method for peptide identification from MS/MS, Proteomics 7, 655–667.
Lam, H., Deutsch, E. W., Eddes, J. S. et al. (2008) Building consensus spectral libraries for peptide identification in proteomics, Nat Methods 5, 873–875.
Fusaro, V. A., Mani, D. R., Mesirov, J. P., and Carr, S. A. (2009) Prediction of high-responding peptides for targeted protein assays by mass spectrometry, Nat Biotechnol 27, 190–198.
Schmidt, A., Claassen, M., and Aebersold, R. (2009) Directed mass spectrometry: towards hypothesis-driven proteomics, Curr Opin Chem Biol 13, 510–517.
Keller, A., Eng, J., Zhang, N., Li, X. J., and Aebersold, R. (2005) A uniform proteomics MS/MS analysis platform utilizing open XML file formats, Mol Syst Biol 1, 2005 0017.
Malmstrom, J., Beck, M., Schmidt, A. et al. (2009) Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans, Nature 460, 762–765.
Kitteringham, N. R., Jenkins, R. E., Lane, C. S. et al. (2009) Multiple reaction monitoring for quantitative biomarker analysis in proteomics and metabolomics, J Chromatogr B Analyt Technol Biomed Life Sci 877, 1229–1239.
Pan, S., Aebersold, R., Chen, R. et al. (2009) Mass spectrometry based targeted protein quantification: methods and applications, J Proteome Res 8, 787–797.
Elias, J. E., and Gygi, S. P. (2007) Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry, Nat Methods 4, 207–214.
Reiter, L., Rinner, O., Picotti, P., et al. (2011) mProphet: A general and flexible data model and algorithm for automated SRM data processing and statistical error estimation, Nat Methods, submitted.
Sherwood, C. A., Eastham, A., Lee, L. W. et al. (2009) Rapid optimization of MRM-MS instrument parameters by subtle alteration of precursor and product m/z targets, J Proteome Res 8, 3746–3751.
MacLean, B., Tomazela, D. M., Shulman, N. et al. (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments, Bioinformatics 26, 966–968.
Sherwood, C. A., Eastham, A., Lee, L. W. et al. (2009) MaRiMba: a software application for spectral library-based MRM transition list assembly, J Proteome Res 8, 4396–4405.
Sherman, J., McKay, M. J., Ashman, K., and Molloy, M. P. (2009) How specific is my SRM?: The issue of precursor and product ion redundancy, Proteomics 9, 1120-1123.
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Ossola, R., Schiess, R., Picotti, P., Rinner, O., Reiter, L., Aebersold, R. (2011). Biomarker Validation in Blood Specimens by Selected Reaction Monitoring Mass Spectrometry of N-Glycosites. In: Simpson, R., Greening, D. (eds) Serum/Plasma Proteomics. Methods in Molecular Biology, vol 728. Humana Press. https://doi.org/10.1007/978-1-61779-068-3_11
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DOI: https://doi.org/10.1007/978-1-61779-068-3_11
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