Dataset Information

Tandem LysC/trypsin digestion performs efficient in detergent conditions and under constant denaturating environments

ABSTRACT: All shotgun proteomics experiments rely on efficient proteolysis steps for sensitive peptide/protein identification and quantification. Continuous protocol improvement is therefore a constant topic in proteomics research. Previous reports suggest that the sequential tandem LysC/trypsin digest yields higher recovery of fully tryptic peptides than single-tryptic proteolysis. Based on the previous studies, it is assumed that the advantageous effect of tandem proteolysis requires a high sample denaturation state for the initial LysC digest. Therefore, to date, all systematic assessments of LysC/trypsin proteolysis were done in chaotropic environments such as urea. Here we show that the LysC/trypsin digestion can be carried with high efficiency in MS-compatible detergents resulting in higher yields of fully cleaved peptides. We show that higher cleavage efficiency of tandem digests has an impact on absolute protein quantification using iBAQ values. By additionally testing different urea tandem conditions our data implies that beneficial effects of the initial LysC digest do not depend on the sample denaturation state, but more likely, are due to different target specificities of LysC and trypsin. The observed detergent compatibility enables tandem digestion schemes to be implemented in efficient cellular solubilization proteomics procedures without the need for buffer exchange to chaotropic digestion environment.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli Bacteria

TISSUE(S): Cell Culture

SUBMITTER: Timo Glatter

LAB HEAD: Timo Glatter

PROVIDER: PXD013273 | Pride | 2020-04-30

REPOSITORIES: Pride

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Efficient Tandem LysC/Trypsin Digestion in Detergent Conditions.

Hakobyan Anna A Schneider Martin Bernd MB Liesack Werner W Glatter Timo T

Proteomics 20190930 20

All shotgun proteomics experiments rely on efficient proteolysis steps for sensitive peptide/protein identification and quantification. Previous reports suggest that the sequential tandem LysC/trypsin digest yields higher recovery of fully tryptic peptides than single-tryptic proteolysis. Based on the previous studies, it is assumed that the advantageous effect of tandem proteolysis requires a high sample denaturation state for the initial LysC digest. Therefore, to date, all systematic assessme ...[more]

PMID: 31536157

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