Abstract
We demonstrate the quantification capability and robustness of a new integrated liquid chromatography-mass spectrometry (LC-MS) approach for large-scale profiling of proteins and metabolites. This approach to determine differential expression relies on linearity of signal vs molecular concentration using electrospray ionization LC-MS, reproducibility of sample processing, a novel normalization strategy and associated data analysis software. No isotopic tagging or spiking of internal standards is required. The method is general and applicable to the proteome and metabolome from all biological fluids and tissues. Small or large numbers of samples can be profiled in a single experiment. Differential profiling of 6000 molecular ions per sample by one-dimensional chromatography LC-MS and 30,000 molecular ions per sample by two-dimensional chromatography LC-MS is demonstrated using rheumatoid arthritis patient samples compared with control samples. A new approach to peptide identification is described that involves building libraries of previously identified peptides, circumventing the need to acquire MS/MS data during profiling. Robustness of the platform was tested by repeating sample preparation and LC-MS differential expression analysis after 10 mo, using independent serum aliquots stored at − 80°C. To the best of our knowledge, this is the first demonstration of long-term robustness of a platform for quantitative proteomics and metabolomics.
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Bodovitz, S. and Patterson, S. D. (2003) Protein biomarker strategies. Drug Discovery World Fall 2003, 67–78.
Rubenstein, K. (2003) Post-genomic biomarkers: revolutionizing drug development and diagnostics. D and MD Publications, Westborough, MA.
Wang, W., Zhou, H., Lin, H., et al. (2003) Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards. Anal. Chem. 75, 4818–4826.
Steen, H., Jebanathirajah, J. A., Springer, M., and Kirschner, M. W. (2005) Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS. Proc. Natl. Acad. Sci. USA 102, 3948–3953.
Anderson, N. L. and Anderson, N. G. (2003) The human plasma proteome: history, character and diagnostic prospects. Mol. Cell. Proteomics 2, 50.
Adkins, J. N., Varnum, S. M., Auberry, K. J., et al. (2002) Toward a human blood serum proteome: Analysis by multidimensional separation coupled with mass spectrometry. Mol. Cell. Proteomics 1, 947–955.
Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol. 17, 994–999.
Ji, J., Chakraborty, A., Geng, M., et al. (2000) Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. J. Chromatogr. B 745, 197–210.
Goshe, M. B. and Smith, R. D. (2003) Stable isotope-coded proteomic mass spectrometry. Curr. Opin. Biotechnol. 14, 101–109.
Gerber, S. A., Rush, J., Stemman, O., Kirschner, M. W., and Gygi, S. P. (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. Sci. USA 100, 12,6940–12,6945.
US Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories, 4th Edition, HHS Publication No. (CDC) 93-8395.
Hastings C. A., Norton, S. M., and Roy, S. (2002) New algorithms for processing and peak detection in liquid chromatography/mass spectrometry data. Rapid Commun. Mass Specrom. 16, 462–467.
Anderle, M., Roy, S., Lin, H., Becker, C., and Joho, K. (2004) Quantifying reproducibility for differential proteomics: noise analysis for protein liquid chromatography-mass spectrometry in human serum. Bioinformatics 20, 183,575–183,582.
Roy, S., Anderle, M., Lin, H., and Becker, C. (2004) Differential expression profiling of serum proteins and metabolites for Biomarker Discovery. Int. J Mass Spec. 238, 163–171.
Rabiner, L. and Juang, B.-H. (1993) Fundamentals of Speech Recognition, Prentice-Hall Publishers, Englewood Cliffs, NJ.
Glantz, S. A. (1997) Primer of Biostatistics, McGraw-Hill, New York.
Carr, S., Aebersold, R., Baldwin, M., Burlingame, A., Clauser, K., and Nesvizhskii, A. (2004) The need for guidelines in publication of peptide and protein identification data: working group on publication guidelines for peptide and protein identification data. Mol. Cell. Proteomics 3, 531–533.
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Roy, S.M., Becker, C.H. (2007). Quantification of Proteins and Metabolites by Mass Spectrometry Without Isotopic Labeling. In: Sechi, S. (eds) Quantitative Proteomics by Mass Spectrometry. Methods in Molecular Biology, vol 359. Humana Press. https://doi.org/10.1007/978-1-59745-255-7_6
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DOI: https://doi.org/10.1007/978-1-59745-255-7_6
Publisher Name: Humana Press
Print ISBN: 978-1-58829-571-2
Online ISBN: 978-1-59745-255-7
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