The DOI matches its Crossref record; 28 of the 28 references it cites were found in a registry or an index.
Large-Scale Quantitative Assessment of Different In-Solution Protein Digestion Protocols Reveals Superior Cleavage Efficiency of Tandem Lys-C/Trypsin Proteolysis over Trypsin Digestion
Abstract
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 ensure 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.
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Schmidt, A., Kochanowski, K., Vedelaar, S., Ahrné, E., Volkmer, B., Callipo, L., Knoops, K., Bauer, M., Aebersold, R., & Heinemann, M. (2016) · Nature Biotechnology · doi:10.1038/nbt.3418 · Cited by 771
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“In addition to reducing sample complexity, for accurate and reliable quantification of proteins by targeted analysis, complete and consistent recovery of peptides from the sample is crucial [62,63].”
BackgroundGuo, T., Kouvonen, P., Koh, C. C., Gillet, L. C., Wolski, W. E., Röst, H. L., Rosenberger, G., Collins, B. C., Blum, L. C., Gillessen, S., et al. (2015) · Nature Medicine · doi:10.1038/nm.3807 · Cited by 376
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Correia, A. L., Guimaraes, J. C., Auf der Maur, P., De Silva, D., Trefny, M. P., Okamoto, R., Bruno, S., Schmidt, A., Mertz, K., Volkmann, K., et al. (2021) · Nature · doi:10.1038/s41586-021-03614-z · Cited by 339
Self-citationLarge-scale identification of ubiquitination sites by mass spectrometry
Udeshi, N. D., Mertins, P., Svinkina, T., & Carr, S. A. (2013) · Nature Protocols · doi:10.1038/nprot.2013.120 · Cited by 296
Metadata
- Authors
- Glatter, T., Ludwig, C., Ahrné, E., Aebersold, R., Heck, A. J. R., & Schmidt, A.
- Venue
- Journal of Proteome Research
- Publisher
- American Chemical Society (ACS)
- Published
- 2012-10-16
- Volume
- 11
- Issue
- 11
- Pages
- 5145-5156
- ISSN
- 1535-3893 (ISSN-L)
- 1535-3907
- OpenAlex
- W1997528669
- Cited by
- 364
- References
- 28
- FWCI
- 11.17
- Type
- journal article
- Language
- en
- Open access
- closed
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- Glatter, T., Ludwig, C., Ahrné, E., Aebersold, R., Heck, A. J. R., & Schmidt, A. (2012). Large-Scale Quantitative Assessment of Different In-Solution Protein Digestion Protocols Reveals Superior Cleavage Efficiency of Tandem Lys-C/Trypsin Proteolysis over Trypsin Digestion. Journal of Proteome Research. https://doi.org/10.1021/pr300273g
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