Abstract
A great variety of technologies using stable isotope labeling in combination with mass spectrometry have been described being tools to identify and relatively quantify proteins within complex mixtures. Here, we present a method, termed isotope-coded protein label (ICPL), which is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on tagging stable isotope derivatives at the free amino groups of intact proteins, the method is applicable to any protein sample, including extracts from tissues or body fluids. All separation methods currently employed in proteome studies can be used to reduce complexity on the protein level. After enzymatic cleavage of the protein fractions, the ratios of peptides from different proteome states can be calculated by simple MS-based mass spectrometric analyses. Only peptides representing different expression levels in the different proteomic states are further analyzed by tandem-mass spectrometry to identify respective proteins. For quantification of proteins from multiplexed ICPL experiments, ICPLQuant was developed, a software package especially designed to cover the whole ICPL workflow. The ICPL method results in accurate and reproducible quantification of proteins and high sequence coverage, indispensable for a comprehensive detection of posttranslational modifications and discrimination of protein isoforms.
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References
Ross PL, Huang YN, Marchese JN et al (2004) Multiplexed protein quantitation in SaccharoÂmyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3(12):1154–1169
Kellermann J (2008) Sample preparation for differential proteome analysis: labelling technologies for mass spectrometry. In: von Hagen J (ed) Proteomics sample preparation. Wiley, Weinheim
Lottspeich F, Kellermann J (2011) ICPL labeling strategies for proteome research. Methods Mol Biol 753:55–64
Gygi SP, Rist B, Gerber SA et al (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17:994–999
Ong SE, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1(5):376–386
Schmidt A, Kellermann J, Lottspeich F (2005) A novel strategy for quantitative proteomics using isotope-coded protein labels. Proteomics 5:4–15
Brunner A, Keidel E, Dosch D et al (2010) ICPLQuant—a software for non-isobaric isotopic labeling proteomics. Proteomics 10:315–323
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Kellermann, J., Lottspeich, F. (2012). Isotope-Coded Protein Label. In: Marcus, K. (eds) Quantitative Methods in Proteomics. Methods in Molecular Biology, vol 893. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-885-6_11
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DOI: https://doi.org/10.1007/978-1-61779-885-6_11
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-884-9
Online ISBN: 978-1-61779-885-6
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