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Nat Protoc. 2008;3(9):1444-51. doi: 10.1038/nport.2008.132.

Calculating absolute and relative protein abundance from mass spectrometry-based protein expression data.

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Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, 2500 Speedway, MBB 3.210, Austin, Texas 78712, USA.


Mass spectrometry (MS)-based shotgun proteomics allows protein identifications even in complex biological samples. Protein abundances can then be estimated from the counts of tandem MS (MS/MS) spectra attributable to each protein, provided one accounts for differential MS detectability of contributing peptides. We developed a method, APEX, which calculates Absolute Protein EXpression levels based upon learned correction factors, MS/MS spectral counts and each protein's probability of correct identification. This protocol describes APEX-based calculations in three parts. (i) Using training data, peptide sequences and their sequence properties, a model is built to estimate MS detectability (O(i)) for any given protein. (ii) Absolute protein abundances are calculated from spectral counts, identification probabilities and the learned O(i)-values. (iii) Simple statistics allow calculation of differential expression in two distinct biological samples, i.e., measuring relative protein abundances. APEX-based protein abundances span 3-4 orders of magnitude and are applicable to mixtures of 100s to 1,000s of proteins.

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