Document Detail


Large-scale quantitative assessment of different in-solution protein digestion protocols reveals superior cleavage efficiency of tandem Lys-C/trypsin proteolysis over trypsin digestion.
MedLine Citation:
PMID:  23017020     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
The complete and specific proteolytic cleavage of protein samples into peptides is crucial for the success of every shotgun LC-MS/MS experiment. In particular, popular peptide based label-free and targeted mass spectrometry approaches rely on efficient generation of fully cleaved peptides to assure accurate and sensitive protein quantification. In contrast to previous studies, we globally and quantitatively assessed the efficiency of different digestion strategies using a yeast cell lysate, label-free quantification and statistical analysis. Digestion conditions include double tryptic, surfactant-assisted and tandem-combinatorial Lys-C/trypsin digestion. In comparison to tryptic digests, Lys-C/trypsin digests were found most efficient to yield fully cleaved peptides while reducing the abundance of miscleaved peptides. Subsequent sequence context analysis revealed improved digestion performances of Lys-C/trypsin for miscleaved sequence stretches flanked by charged basic and particulary acidic residues. Furthermore, targeted MS analysis demonstrated a more comprehensive protein cleavage only after Lys-C/trypsin digestion, resulting in a more accurrate absolute protein quantification and extending the number of peptides suitable for SRM assay development. Therefore, we conclude that a serial Lys-C/trypsin digestion is highly attractive for most applications in quantitative MS based proteomics building on in-solution digestion schemes.
Authors:
Timo Glatter; Christina Ludwig; Erik Ahrne; Ruedi Aebersold; Albert J R Heck; Alexander Schmidt
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-9-28
Journal Detail:
Title:  Journal of proteome research     Volume:  -     ISSN:  1535-3907     ISO Abbreviation:  J. Proteome Res.     Publication Date:  2012 Sep 
Date Detail:
Created Date:  2012-9-28     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101128775     Medline TA:  J Proteome Res     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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