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.
Department of Biology, Center for Genomics and Systems Biology, New York University, New York, NY, USA.
Christine
C
Vogel
Edward M
EM
Marcotte
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D002457:Cell Extracts / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods*; D020543:Proteome / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics; D012984:Software; D053719:Tandem Mass Spectrometry
D002457:Cell Extracts; D005656:Fungal Proteins; D010446:Peptide Fragments; D020543:Proteome