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J Proteome Res. 2016 Nov 4;15(11):3988-3997. Epub 2016 Aug 29.

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

Wei W1, Luo W2, Wu F1,3, Peng X1,4, Zhang Y1,5, Zhang M1, Zhao Y1, Su N1, Qi Y1, Chen L1, Zhang Y1, Wen B6, He F1, Xu P1,2,4.

Author information

1
State Key Laboratory of Proteomics, National Center for Protein Sciences Beijing, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, Beijing Institute of Radiation Medicine , Beijing 102206, China.
2
Graduate School, Anhui Medical University , Hefei 230032, China.
3
Life Science College, Southwest Forestry University , Kunming, 650224, China.
4
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of the Ministry of Education, School of Pharmaceutical Sciences, Wuhan University , Wuhan 430072, China.
5
Institute of Microbiology , Chinese Academy of Science, Beijing 100101, China.
6
BGI-Shenzhen , Shenzhen 518083, China.

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.

KEYWORDS:

Chromosome-Centric Human Proteome Project; LTQ Orbitrap Velos; Q-Exactive HF; missing proteins; testis

PMID:
27535590
DOI:
10.1021/acs.jproteome.6b00390
[Indexed for MEDLINE]

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