Abstract

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 (Oi) for any given protein. (ii) Absolute protein abundances are calculated from spectral counts, identification probabilities and the learned Oi-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.