Abstract
A protein molecule exists as a heterogeneous population of posttranslationally modified forms, which are of potential interest to biologists. However, due to detection or methodology limitations, they remain uncharacterized. When a protein does become a prioritized interest in a laboratory, workflows aimed for its purification and characterization are implemented. Inherent in these workflows is the enrichment of the protein from the biological lysate, rendering it an ideal sample for mass spectrometry (MS), as detection of several peptides is greatly increased. In order to capitalize on this enhanced detection of the protein of interest, we have developed a full-length expressed protein quantification standard (FLEXIQuant standard) that is in vitro synthesized, devoid of posttranslational modifications (PTMs), and implemented into the purification workflow of the endogenous counterpart—as such it serves as an internal MS standard. FLEXIQuantification allows for the unbiased identification of peptides undergoing PTM as a function of a particular biological state. The extent of PTM is also quantified, providing further insight into the regulation of the protein.
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References
Xu P, Duong DM, Seyfried NT et al (2009) Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137(1):133–145
Steen H, Jebanathirajah JA, Springer M, Kirschner MW (2005) Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS. Proc Natl Acad Sci USA 102(11):3948–3953
Steen JA, Steen H, Georgi A et al (2008) Different phosphorylation states of the anaphase promoting complex in response to antimitotic drugs: a quantitative proteomic analysis. Proc Natl Acad Sci USA 105(16):6069–6074
Olsen JV, Blagoev B, Gnad F et al (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127(3):635–648
Zhang Y, Wolf-Yadlin A, Ross PL et al (2005) Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules. Mol Cell Proteomics 4(9):1240–1250
Gygi SP, Rist B, Gerber SA et al (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17(10):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
Gerber SA, Rush J, Stemman O et al (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci USA 100(12):6940–6945
Singh S, Springer M, Steen J et al (2009) FLEXIQuant: a novel tool for the absolute quantification of proteins, and the simultaneous identification and quantification of potentially modified peptides. J Proteome Res 8(5):2201–2210
Schmidt A, Kellermann J, Lottspeich F (2005) A novel strategy for quantitative proteomics using isotope-coded protein labels. Proteomics 5(1):4–15
Brun V, Dupuis A, Adrait A et al (2007) Isotope-labeled protein standards: toward absolute quantitative proteomics. Mol Cell Proteomics 6(12):2139–2149
Janecki DJ, Bemis KG, Tegeler TJ et al (2007) A multiple reaction monitoring method for absolute quantification of the human liver alcohol dehydrogenase ADH1C1 isoenzyme. Anal Biochem 369(1):18–26
Goshima N, Kawamura Y, Fukumoto A et al (2008) Human protein factory for converting the transcriptome into an in vitro-expressed proteome. Nat Methods 5(12):1011–1017
Kall L, Storey JD, MacCoss MJ, Noble WS (2008) Assigning significance to peptides identified by tandem mass spectrometry using decoy databases. J Proteome Res 7(1):29–34
Kapust RB, Tozser J, Copeland TD, Waugh DS (2002) The P1′ specificity of tobacco etch virus protease. Biochem Biophys Res Commun 294(5):949–955
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Singh, S., Kirchner, M., Steen, J.A., Steen, H. (2012). A Practical Guide to the FLEXIQuant Method. 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_19
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DOI: https://doi.org/10.1007/978-1-61779-885-6_19
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