Absolute protein quantification is an essential tool for system biology approaches and elucidation of stoichiometry of multi-protein complexes. In this chapter, a universal protocol for gel free absolute protein quantification in bacterial systems is described, which can be used for sample preparation prior to miscellaneous mass-spectrometry-based quantification workflows like AQUA, Hi3, and emPAI. In addition, a focus has been set to the specific challenges in antibiotic stress research.
Gel free proteomics Sample preparation Absolute protein quantification In-solution digestion AQUA QconCAT Hi3 emPAI Antibiotic stress response
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
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 100(12):6940–6945CrossRefPubMedPubMedCentralGoogle Scholar
Pratt JM, Simpson DM, Doherty MK et al (2006) Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes. Nat Protoc 1(2):1029–1043CrossRefPubMedGoogle Scholar
Maass S, Sievers S, Zühlke D et al (2011) Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics. Anal Chem 83(7):2677–2684CrossRefPubMedGoogle Scholar
Ishihama Y, Odat Y, Tabata T et al (2005) Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 4(9):1265–1272CrossRefPubMedGoogle Scholar
Schwanhäusser B, Busse D, Li N et al (2011) Global quantification of mammalian gene expression control. Nature 473(7347):337–342CrossRefPubMedGoogle Scholar
Silva JC, Gorenstein Marc V, Guo-Zhong L et al (2006) Absolute quantification of proteins by LCMSE. Mol Cell Proteomics 5(1):144–156CrossRefPubMedGoogle Scholar