Automated protein (re)sequencing with MS/MS and a homologous database yields almost full coverage and accuracy.

Abstract

The bottom-up tandem mass spectrometry (MS/MS) is regularly used in proteomics nowadays for identifying proteins from a sequence database. De novo sequencing software is also available for sequencing novel peptides with relatively short sequence lengths. However, automated sequencing of novel proteins from MS/MS remains a challenging problem. Very often, although the target protein is novel, it has a homologous protein included in a known database. When this happens, we propose a novel algorithm and automated software tool, named Champs, for sequencing the complete protein from MS/MS data of a few enzymatic digestions of the purified protein. Validation with two standard proteins showed that our automated method yields >99% sequence coverage and 100% sequence accuracy on these two proteins. Our method is useful to sequence novel proteins or 're-sequence' a protein that has mutations comparing with the database protein sequence.