Decoding the Effect of Isobaric Substitutions on Identifying Missing Proteins and Variant Peptides in Human Proteome

To
confirm the existence of missing proteins, we need to identify
at least two unique peptides with length of 9–40 amino acids
of a missing protein in bottom-up mass-spectrometry-based proteomic
experiments. However, an identified unique peptide of the missing
protein, even identified with high level of confidence, could possibly
coincide with a peptide of a commonly observed protein due to isobaric
substitutions, mass modifications, alternative splice isoforms, or
single amino acid variants (SAAVs). Besides unique peptides of missing
proteins, identified variant peptides (SAAV-containing peptides) could
also alternatively map to peptides of other proteins due to the aforementioned
issues. Therefore, we conducted a thorough comparative analysis on
data sets in PeptideAtlas Tiered Human Integrated Search Proteome
(THISP, 2017-03 release), including neXtProt (2017-01 release), to
systematically investigate the possibility of unique peptides in missing
proteins (PE2–4), unique peptides in dubious proteins, and
variant peptides affected by isobaric substitutions, causing doubtful
identification results. In this study, we considered 11 isobaric substitutions.
From our analysis, we found <5% of the unique peptides of missing
proteins and >6% of variant peptides became shared with peptides
of
PE1 proteins after isobaric substitutions.