Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues

Abstract

Almost all large-scale projects in mass spectrometrybased 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. Molecular & Cellular Proteomics 3:608 -614, 2004. Mass spectrometry (MS)1 -based proteomics almost invariably involves the enzymatic degradation of proteins to peptides by trypsin (1). This protease has high cleavage specificity, is very aggressive, and is stable under a wide variety of conditions. Most importantly, cleaving C-terminal to arginine or lysine residues leads to peptides in the preferred mass range for effective fragmentation by tandem mass spectrometry (MS/MS) and places the highly basic residues at the C termini of the peptides. This generally leads to informative high mass y-ion series and makes tandem mass spectra more easily interpretable.When analyzing peptide mixtures by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), a large number of fragmentation events occur. The tandem mass spectra are searched against amino acid sequence databases by one of a number of database search algorithms. The identified peptides receive a score and are combined into lists of identified proteins. A critical question in these experiments is what constitutes a reliable peptide and protein hit (2). Some laboratories save raw mass spectrometric data and interpret this raw data in all questionable cases. In some algorithms, the score is itself a probability and can be used to estimate levels of false positives (incorrect hits) and false negatives (missed hits). For other algorithms, this question has been addressed by analyzing defined mixtures of known proteins (3); or by searching in reversed databases that should not yield significant hits (4, 5). On the basis of these findings, a set of parameters for the scores is often defined that will yield a given trade-off of false positives and false negatives. Recently, more sophisticated statistical learning algorithms have been...

Analytical characterization of NOTA-modified somatropins

“…8.0: 01/2008:0951) [58]. It is commonly known that trypsin is a specific protease that cleaves at the c-terminal of arginine and lysine residues [60]. This yields theoretically 21 peptide fragments for somatropin (PeptideCutter [61]).…”

A proteomic study of the HUPO Plasma Proteome Project's pilot samples using an accurate mass and time tag strategy

“…Considerable research effort has been directed at determining appropriate data filters, protease cleavage states, mass measurement accuracy, and the number of peptide identifications required for a confident protein identification [15,17,27,32,[46][47][48][49][50]. Blood serum and plasma are increasingly proving to be a more difficult to fully characterize with traditional proteomic technologies, as shown by both the HUPO and other efforts.…”

“…One school of thought has been that nearly all peptides result from highly specific digestion by the exogenous protease, typically trypsin, and thus all confident peptide identifications should conform to fully tryptic digestion patterns (e.g., [10,48]). Alternatively, complex protein mixtures processed by endogenous proteases may contain unexpected amino-and carboxy-termini, resulting in non-tryptic cleavage states (e.g., [15,18,27]).…”

Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins

“…The use of accurate mass measurements in combination with HPLC, spawned novel methods for database searching such as the use of accurate mass tags and retention times to create databases, which can then be used for identification and quantitation purposes [17]. In addition, the recent ease of use and ability to perform high throughput bottom-up LCMS experiments using the ICR cell as a mass detector have facilitated the implementation of these measurements for routine experiments involving peptide sequencing for protein identification purposes [18]. This has given rise to another original method of determining the sequence of a peptide where accurate masses of fragment ions can be used to determine elemental composition and, hence, amino acid composition, composition based sequencing (CBS), which can be carried out, i.e., for de novo sequencing purposes [19,20].…”