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Mol Cell Proteomics. 2014 Mar;13(3):823-35. doi: 10.1074/mcp.M113.034710. Epub 2014 Jan 14.

Expanding proteome coverage with orthogonal-specificity α-lytic proteases.

Author information

1
Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Dr., La Jolla, California 92093-0378;

Abstract

Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage through cleavage at sequences complementary to trypsin's may increase proteome coverage. We demonstrate the novel application of two proteases for bottom-up proteomics: wild type α-lytic protease (WaLP) and an active site mutant of WaLP, M190A α-lytic protease (MaLP). We assess several relevant factors, including MS/MS fragmentation, peptide length, peptide yield, and protease specificity. When data from separate digestions with trypsin, LysC, WaLP, and MaLP were combined, proteome coverage was increased by 101% relative to that achieved with trypsin digestion alone. To demonstrate how the gained sequence coverage can yield additional post-translational modification information, we show the identification of a number of novel phosphorylation sites in the Schizosaccharomyces pombe proteome and include an illustrative example from the protein MPD2 wherein two novel sites are identified, one in a tryptic peptide too short to identify and the other in a sequence devoid of tryptic sites. The specificity of WaLP and MaLP for aliphatic amino acid side chains was particularly valuable for coverage of membrane protein sequences, which increased 350% when the data from trypsin, LysC, WaLP, and MaLP were combined.

PMID:
24425750
PMCID:
PMC3945911
DOI:
10.1074/mcp.M113.034710
[Indexed for MEDLINE]
Free PMC Article

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