Label-free protein quantitation using weighted spectral counting

Mass spectrometry (MS)-based shotgun proteomics allows protein identifications even in complex biological samples. Protein abundances can then be estimated from the counts of MS/MS spectra attributable to each protein, provided that one corrects for differential MS-detectability of the contributing peptides. We describe the use of a method, APEX, which calculates Absolute Protein EXpression levels based on learned correction factors, MS/MS spectral counts, and each protein's probability of correct identification. The APEX-based calculations consist of three parts: (1) Using training data, peptide sequences and their sequence properties, a model is built that can be used to estimate MS-detectability (O _i) for any given protein. (2) Absolute abundances of proteins measured in an MS/MS experiment are calculated with information from spectral counts, identification probabilities and the learned O _i-values. (3) Simple statistics allow for significance analysis of differential expression in two distinct biological samples, i.e., measuring relative protein abundances. APEX-based protein abundances span more than four orders of magnitude and are applicable to mixtures of hundreds to thousands of proteins from any type of organism.