Limitations of Mass Spectrometry-Based Peptidomic Approaches

Mass spectrometry-based peptidomic approaches are powerful techniques to detect and identify the peptide content of biological samples. The present study investigated the limitations of peptidomic approaches using trimethylammonium butyrate isotopic tags to quantify relative peptide levels and Mascot searches to identify peptides. Data were combined from previous studies on human cell lines or mouse tissues. The combined databases contain 2155 unique peptides ranging in mass from 444 to 8765 Da, with the vast majority between 1 and 3 kDa. The amino acid composition of the identified peptides generally reflected the frequency in the Eukaryotic proteome with the exception of Cys, which was not present in any of the identified peptides in the free-SH form but was detected at low frequency as a disulfide with Cys residues, a disulfide with glutathione, or as S-cyanocysteine. To test if the low detection rate of peptides smaller than 500 Da, larger than 3 kDa, or containing Cys was a limitation of the peptidomics procedure, tryptic peptides of known proteins were processed for peptidomics using the same approach used for human cell lines and mouse tissues. The identified tryptic peptides ranged from 516 to 2418 Da, whereas the theoretical digest ranged from 217 to 7559 Da. Peptides with Cys were rarely detected and, if present, the Cys was usually modified S-cyanocysteine. Additionally, peptides with mono- and di-iodo Tyr and His were identified. Taken together, there are limitations of peptidomic techniques, and awareness of these limitations is important to properly use and interpret results. Graphical Abstract ᅟ.