Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues*S

By Jesper V. Olsen, Shao-En Ong, Matthias Mann. Molecular & Cellular Proteomics, 2004

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Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. Here we use the sub-parts per million mass accuracy of a new ion trap Fourier transform mass spectrometer to achieve more than a 100-fold increased confidence in peptide identification compared with typical ion trap experiments and show that trypsin cleaves solely C-terminal to arginine and lysine. We find that non-tryptic peptides occur only as the C-terminal peptides of proteins and as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins erroneously identified with non-tryptic peptide hits. Our results indicate that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity.

Subfields: Molecular Biology, Spectroscopy

View Paper: https://www.semanticscholar.org/paper/0c8b5c7af043b6ed65711a31ea4c76e72546eaf5

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Citations in the corpus

A Proteome-wide, Quantitative Survey of In Vivo Ubiquitylation Sites Reveals Widespread Regulatory Roles (2011, background): “The strict sequence specificity of trypsin (38), and its inability to cleave at ubiquitylated lysines greatly facilitated the unambiguous localization of ubiquitylation sites.”
Phosphotyrosine interactome of the ErbB-receptor kinase family (2005, methodology): “…peptides that were then searched against the human International Protein Index Database (IPI; http://srs6.ebi.ac.uk) using the Mascot algorithm (Perkins et al, 1999) with search parameters as described in (Foster et al, 2003) for QSTAR data and as in Olsen et al (2004) for LTQ-FT data.”
Mass spectrometry–based proteomics turns quantitative (2005, background): “) The dominant sample analysis workflow in proteomics utilizes site-specific enzymatic proteases such as trypsin to digest proteins to peptide”
Parts per Million Mass Accuracy on an Orbitrap Mass Spectrometer via Lock Mass Injection into a C-trap*S (2005, methodology): “However, here we achieve the high mass accuracy with the help of a lock mass whereas in the LTQ-FTICR we used “SIM scans,” the isolation and injection of a narrow mass range into the ICR cell ( 3 ).”
Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures (2009, background): “Other reports imply however that enzymes are specific and reproducible and as such use stringent enzyme specificity [24, 25].”
A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC) (2006, background): “Trypsin is a common proteolytic enzyme in proteomics workflows because it is a very aggressive yet specific protease that cleaves at the carboxyl-termini of lysine and arginine residue”
Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers. (2010, methodology): “Peptide identification was performed on an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Germany) essentially as described (Olsen et al., 2004).”
SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function. (2008, background): “collisionally induced dissociation at a target value of 10,000 (Olsen et al., 2004).”