Limitations of Mass Spectrometry-Based Peptidomic Approaches

Abstract

Abstract. 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.

Analysis of the Yeast Peptidome and Comparison with the Human Peptidome

“…The amino acid composition of the peptides shows the most abundant amino acid is Ala (10.6%), with Lys, Val, Leu, Gly, Asp, and Glu also abundant ( Table 1 ). Cys is not detected in the peptidome, but this omission is likely to be an artifact of the extraction and/or labeling procedure [ 43 ]. Otherwise, the amino acid composition of the peptidome is generally similar to the amino acid composition of the intracellular and nuclear yeast proteome [ 44 ].…”

“…A previous analysis of intracellular mouse brain peptides found that close to one half of the peptides represented the N- or C-terminus of the protein [ 16 ]. In contrast, proteomic studies that digest proteins with trypsin prior to MS analysis detect mainly peptides that are internal fragments of the protein [ 43 ]. Similar to the previous report on intracellular mouse peptides, 43% of the human cell line peptides represent either the N-terminus (26%) or C-terminus (17%) of the proteins from which the peptides are derived ( Fig 2 ).…”

ProSAAS‐derived peptides are regulated by cocaine and are required for sensitization to the locomotor effects of cocaine

“…With caveats, such as the need to heat‐stabilize brain tissue prior to peptide extraction, the quantitative peptidomics technique used in this study is relatively unbiased and permits the detection of a large number of neuropeptides and other peptides in brain tissue (Fricker ). The proSAAS‐derived peptides are the most commonly detected peptides in peptidomic studies, consistent with the high abundance of these peptides in brain (Fricker ).…”

Biological activities and peptidomic profile of in vitro-digested cow, camel, goat and sheep milk

“…The mass spectrometer was tuned, calibrated and set with the same parameters as reported by Dei Più et al (2014). This approach suffers of several limitations especially related to the detection and identification of small peptides and any peptide containing free cysteine (Fricker, 2015). Small peptides (<500 Da) are often inefficiently ionized giving a low intensity m/z signal which hampered the selection of precursor for successive MS/MS fragmentation.…”