Improved Protein Inference from Multiple Protease Bottom-Up Mass Spectrometry Data.

Peptides detected by tandem mass spectrometry (MS/MS) in bottom-up proteomics serve as proxies for the proteins expressed in the sample. Protein inference is a process routinely applied to these peptides to generate a plausible list of candidate protein identifications. The use of multiple proteases for parallel protein digestions expands sequence coverage, provides additional peptide identifications, and increases the probability of identifying peptides that are unique to a single protein, which are all valuable for protein inference. We have developed and implemented a multi-protease protein inference algorithm in MetaMorpheus, a bottom-up search software program, which incorporates the calculation of protease-specific q-values and preserves the association of peptide sequences and their protease of origin. This integrated multi-protease protein inference algorithm provides more accurate results than the aggregation of results from the separate analysis of the peptide identifications produced by each protease (separate approach) in MetaMorpheus, and results obtained using either Fido, ProteinProphet or DTASelect2. MetaMorpheus and its integrated multi-protease data analysis (integrated approach) decreases the ambiguity of the protein group list, reduces the frequency of erroneous identifications, and increases the number of post-translational modifications identified while providing increased efficiency by combining multi-protease search and protein inference into a single software program.