Open-pFind Verified Four Missing Proteins from Multi-Tissues

Abstract

The Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2012 to perfect the annotation of human protein existence by identifying stronger evidence of the expression of missing proteins (MPs) at the protein level. After an 8 year effort all over the world, the number of MPs in the neXtProt database significantly decreased from 5511 (2012-02-24) to 1899 (2020-01-17). It is now more difficult to provide confident evidence of the remaining MPs because of their specific characteristics, including low abundance, low molecular weight, unexpected modifications, transmembrane structure, tissue-expression specificity, and so on. A higher resolution mass spectrometry (MS) interpretation engine might provide an opportunity to identify these buried MPs in complex samples by the combination with multi-tissue large-scale proteomics. In this study, open-pFind was used to dig MPs from 20 pairs of healthy human tissues by Wang et al. (Mol. Syst. Biol.201915e8503) combined with our large-scale testis data set digested by three enzymes (Glu-C, Lys-C, and trypsin) with specificity for different amino acid residues (J. Proteme Res.20191841894196). A total of 1 535 536 peptides with 17 283 477 peptide-spectrum matches (PSMs) were mapped to 14 279 protein entries at a false discovery rate of <1% at the PSM, peptide, and protein levels. A total of 103 MP candidates were identified, among which 86 candidates had more unique peptide numbers compared with our single testis tissue. After rigorous screening, manual checks, peptide synthesis, and matching with documented peptides from PeptideAtlas, we validated four MPs, P0C7T8 (duodenum and small intestine), Q8WWZ4 (stomach and rectum), Q8IV35 (fallopian tube), and O14921 (tonsil), at the protein level. All MS raw files have been deposited to the ProteomeXchange with identifier PXD021391.

dbPTM in 2022: an updated database for exploring regulatory networks and functional associations of protein post-translational modifications

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A Massive Proteogenomic Screen Identifies Thousands of Novel Peptides From the Human “Dark” Proteome

“…In the third MS run, 5 out of 10 databases were used as controls to make sure that the spectrum matched with novel peptides. For novel peptide/protein selection, our relaxed standard is the common setting for similar studies of novel protein identification ( 21 , 22 , 40 , 58 ). We applied two more stringent filtering criteria (stringent and strictest) and used the program PepQuery to further increase the confidence of some novel peptides/proteins.…”

Interactome Analysis of ClpX Reveals Its Regulatory Role in Metabolism and Photosynthesis in Cyanobacteria

“…The Synechocystis protein database, sourced from the CyanoBase online Web site (http://genome.kazusa.or.jp/cyanobase) 28 was searched for the MS/MS spectra using an open-search algorithm in the pFind software (version 3.1.6). 29 Additionally, a reverse decoy database and common contaminants were used. Trypsin was allowed to undergo two missed cleavages.…”