Deep Coverage Proteomics Identifies More Low-Abundance Missing Proteins in Human Testis Tissue with Q-Exactive HF Mass Spectrometer

Abstract

Since 2012, missing proteins (MPs) investigation has been one of the critical missions of Chromosome-Centric Human Proteome Project (C-HPP) through various biochemical strategies. On the basis of our previous testis MPs study, faster scanning and higher resolution mass-spectrometry-based proteomics might be conducive to MPs exploration, especially for low-abundance proteins. In this study, Q-Exactive HF (HF) was used to survey proteins from the same testis tissues separated by two separating methods (tricine- and glycine-SDS-PAGE), as previously described. A total of 8526 proteins were identified, of which more low-abundance proteins were uniquely detected in HF data but not in our previous LTQ Orbitrap Velos (Velos) reanalysis data. Further transcriptomics analysis showed that these uniquely identified proteins by HF also had lower expression at the mRNA level. Of the 81 total identified MPs, 74 and 39 proteins were listed as MPs in HF and Velos data sets, respectively. Among the above MPs, 47 proteins (43 neXtProt PE2 and 4 PE3) were ranked as confirmed MPs after verifying with the stringent spectra match and isobaric and single amino acid variants filtering. Functional investigation of these 47 MPs revealed that 11 MPs were testis-specific proteins and 7 MPs were involved in spermatogenesis process. Therefore, we concluded that higher scanning speed and resolution of HF might be factors for improving the low-abundance MP identification in future C-HPP studies. All mass-spectrometry data from this study have been deposited in the ProteomeXchange with identifier PXD004092.

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“…This left 525 still missing proteins in testis and spermatozoa. The same teams have continued their analyses and reported further identifications this year, with 47 more in testis from Wei et al 14 and 206 more in spermatozoa from Vandenbrouck et al 15 These teams used SRM and immunohistochemistry to strengthen evidence of their identifications. Wei et al 14 used a fast-scanning, high-resolution Q Exactive HF instrument to detect many low-abundance missing proteins from a total of 8526 proteins that are supported by transcript expression in testis.…”

“…The same teams have continued their analyses and reported further identifications this year, with 47 more in testis from Wei et al 14 and 206 more in spermatozoa from Vandenbrouck et al 15 These teams used SRM and immunohistochemistry to strengthen evidence of their identifications. Wei et al 14 used a fast-scanning, high-resolution Q Exactive HF instrument to detect many low-abundance missing proteins from a total of 8526 proteins that are supported by transcript expression in testis. They identified 81 missing protein candidates, including 15 membrane proteins, 14 testis-specific proteins, and 9 spermatogenesis-related proteins; of these, 47 candidates survived scrutiny of the spectra, the requirements for two uniquely mapping (proteotypic) peptides of at least nine amino acid residues in length, and a search for potential matches to more abundant proteins with single amino acid variants or isobaric PTM modifications.…”

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“…These next-generation mass spectrometers are therefore attractive as they should make possible to obtain more data from the same sample. Indeed, the depth of proteome coverage is increasingly high, and one of the most recently released instruments, the Q Exactive HF tandem mass spectrometer (Thermo), allowed the detection of a set of 8,526 proteins from human tissue (Wei et al, 2016).…”