A Systematic Evaluation of Semispecific Peptide Search Parameter Enables Identification of Previously Undescribed N-Terminal Peptides and Conserved Proteolytic Processing in Cancer Cell Lines

Abstract

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the most commonly used technique in explorative proteomic research. A variety of open-source tools for peptide-spectrum matching have become available. Most analyses of explorative MS data are performed using conventional settings, such as fully specific enzymatic constraints. Here we evaluated the impact of the fragment mass tolerance in combination with the enzymatic constraints on the performance of three search engines. Three open-source search engines (Myrimatch, X! Tandem, and MSGF+) were evaluated concerning the suitability in semi- and unspecific searches as well as the importance of accurate fragment mass spectra in non-specific peptide searches. We then performed a semispecific reanalysis of the published NCI-60 deep proteome data applying the most suited parameters. Semi- and unspecific LC-MS/MS data analyses particularly benefit from accurate fragment mass spectra while this effect is less pronounced for conventional, fully specific peptide-spectrum matching. Search speed differed notably between the three search engines for semi- and non-specific peptide-spectrum matching. Semispecific reanalysis of NCI-60 proteome data revealed hundreds of previously undescribed N-terminal peptides, including cases of proteolytic processing or likely alternative translation start sites, some of which were ubiquitously present in all cell lines of the reanalyzed panel. Highly accurate MS2 fragment data in combination with modern open-source search algorithms enable the confident identification of semispecific peptides from large proteomic datasets. The identification of previously undescribed N-terminal peptides in published studies highlights the potential of future reanalysis and data mining in proteomic datasets.

The protein phosphorylation landscape in photosystem I of the desert algae Chlorella sp.

“…Semi‐specific peptide searches facilitate the detection of endogenous proteolytic activity, often evident in protein degradation processes. Such activity leads to protein cleavage, producing truncated versions with terminal amino acids that may differ from the specificity of the enzyme used during proteomic sample processing (Fahrner et al ., 2021). Surprisingly, LL cells accumulated significantly more cleaved peptides of many PSI and all LHCI subunits (Fig.…”

Targeted and explorative profiling of kallikrein proteases and global proteome biology of pancreatic ductal adenocarcinoma, chronic pancreatitis, and normal pancreas highlights disease-specific proteome remodelling

“…We have previously shown that semi-tryptic (re-)analysis of proteomic data might yield novel insight into endogenous proteolytic processing prior to sample harvesting and eventual trypsination [61] , [62] , [63] . Naturally, this re-analysis can only shed light on non-tryptic, endogenous proteolytic processing.…”

The protein phosphorylation landscape in photosystem I of the desert algae Chlorella sp.

“…Semi-specific peptide searches facilitate the detection of endogenous proteolytic activity, often evident in protein degradation processes. Such activity leads to protein cleavage, producing truncated versions with terminal amino acids that may differ from the specificity of the enzyme used during proteomic sample processing (Fahrner et al ., 2021). In our search for semi-tryptic-cleaved peptides in the PSI of both LL and HL cells as potential indicators of protein degradation, we observed no increase in peptide cleavage for psaD or any other PSI subunit at HL cells (Table S6).…”