Digging for Missing Proteins Using Low-Molecular-Weight Protein Enrichment and a “Mirror Protease” Strategy

Abstract

In 2012, the Chromosome-centric Human Proteome Project (C-HPP) launched an investigation for missing proteins (MPs) to complete the Human Proteome Project (HPP). The majority of the MPs were distributed in lowmolecular-weight (LMW) ranges, especially from 0 to 40 kDa. LMW protein identification is challenging, owing to their short length, low abundance, and hydrophobicity. Furthermore, many sequences from trypsin digestion are unlikely to yield detectable peptides or a reasonable quality of MS 2 spectrum. Therefore, we focused on small MPs by combining LMW protein enrichment and a pair of complementary proteases strategy with trypsin and LysargiNase for human testis samples. In-depth testis LMW protein profiling resulted in the identification of 4063 proteins, of which 2565 were LMW proteins and 1130 had pairs of peptides generated from both trypsin and LysargiNase. This provided additional mass spectral evidence of further verification of small MPs. Finally, two MPs were verified from the seven MP candidates. One of them, Q8N688, was verified with two series of continuous and complementary b/y-product ions from the pairs of spectra for tryptic and LysargiNase digested peptides after the "mirror spectrum" matching. This make the confident identification of the representative peptides for the target MPs. On the contrary, the two verified peptides for Q86WR6 were identified with the same strategy from the gel-separation and gelelution samples, respectively. Although the other five MP candidates showed high-quality spectra, they could not be sufficiently distinguished as PE1s and require further verification. All MS data sets have been deposited in the ProteomeXchange with identifier PXD010093.

Toward Completion of the Human Proteome Parts List: Progress Uncovering Proteins That Are Missing or Have Unknown Function and Developing Analytical Methods

“…One MS candidate was confirmed by finding a matching stranded peptide in PRIDE. Two ranked among the top 10 MS candidates have been reported in this Special Issue, RNF112 by Macron et al 2 in CSF and C17orf64 by He et al 17 in testis, adding further comfort to their confident identification. These 3 found MPs will move Chr 17 to at least 46 MPs found when the neXtProt 2019-01 version is released.…”

“…Of the 14 MPs, 3 were testis-specific proteins and 10 were closely related to liver cancer, potentially useful for biomarker development in hepatocellular carcinoma. He et al 17 used the “mirror protease” approach, whereby the new protease LysargiNase (Huesgen et al), 18 which cleaves N-terminal to lysine and arginine and so mirrors trypsin (cutting C-terminal to lysine and arginine), was used with Tricine-SDS-PAGE enrichment for low-molecular-weight (LMW) proteins and identified 4063 proteins and 2565 LMW (<40 kDa) proteins, of which 1130 had paired peptides generated from the two proteases. Among seven MP candidates, they verified two with a series of continuous and complementary b/y-product ions in the paired spectra: beta-defensin 123 (Q8N688) and C17orf64 (Q86WR6).…”

Trastuzumab-induced upregulation of a protein set in extracellular vesicles emitted by ErbB2-positive breast cancer cells correlates with their trastuzumab sensitivity

“…ITB1 is a transmembrane receptor that mediates cell-ECM adhesion [31], GNAS2 [32] and GNA13 [33] are G proteins bound to transmembrane G protein-coupled receptors, and CTL2 is a transmembrane protein that mediates choline transport [34] while RAB10 is a RAS-related small GTPase [35]. Of note, LC-MS/MS did not detect EV-associated PERP in this model which is not uncommon [36,37] as factors, such as low protein abundance and glycosylation, may limit protein detection in cells and EVs by MS [38,39]. However, importantly, in agreement with the western blotting data based on the cultured cells (Fig.…”

Proteogenomics Study of Blastobotrys adeninivorans TMCC 70007—A Dominant Yeast in the Fermentation Process of Pu-erh Tea

“…The peptides (0.1–1 pmol) were desalted with homemade C18 StageTips and analyzed with LC–MS/MS as described above. A cosine similarity score between the spectrum generated by the synthesized peptide and the corresponding spectrum from the large-scale proteogenomics study was calculated as described previously …”

“…A cosine similarity score between the spectrum generated by the synthesized peptide and the corresponding spectrum from the large-scale proteogenomics study was calculated as described previously. 46 ■ RESULTS The final de novo assembled genome was analyzed to predict protein coding genes using an ab initio algorithm GeneMark-ES. 29,47 A total of 5894 putative protein coding genes with 1463 introns were predicted, and the average gene length is 1514 bp.…”