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J Proteome Res. 2018 Dec 7;17(12):4171-4177. doi: 10.1021/acs.jproteome.8b00397. Epub 2018 Oct 16.

Multiproteases Combined with High-pH Reverse-Phase Separation Strategy Verified Fourteen Missing Proteins in Human Testis Tissue.

Sun J1,2, Shi J1,2, Wang Y2, Chen Y2, Li Y2, Kong D3, Chang L2, Liu F1, Lv Z1, Zhou Y4, He F2, Zhang Y5, Xu P1,2,6.

Author information

1
Hebei Province Key Lab of Research and Application on Microbial Diversity, College of Life Sciences , Hebei University , Baoding , Hebei 071002 , China.
2
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing) , Beijing Institute of Lifeomics , Beijing 102206 , China.
3
Department of Hepatopancreatobiliary Surgery , The Second Affiliated Hospital of Tianjin Medical University , Tianjin 300211 , China.
4
Demo Laboratory of Thermofisher Scientific China , Shanghai 200120 , China.
5
State Key Laboratory of Biocontrol, Guangdong Key Laboratory of Plant Resources, School of Life Sciences , Sun Yat-Sen University , Guangzhou 510275 , China.
6
Key Laboratory of Combinational Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, School of Pharmaceutical Science , Wuhan University , Wuhan 430072 , China.

Abstract

Subsequent to conducting the Chromosome-Centric Human Proteome Project, we have focused on human testis-enriched missing proteins (MPs) since 2015. For protein coverage to be enhanced, a multiprotease strategy was used for separation of samples by 10% SDS-PAGE. For the separating efficiency to be improved, a high-pH reverse phase (RP) separation strategy was applied to fractionate complex samples in this study. A total of 11,558 proteins was identified, which is the largest proteome data set for single human tissue sample so far. On the basis of this large-scale data set, we verified 14 MPs (PE2) in neXtProt (2018-01) after spectrum quality analysis, isobaric post-translational modification, and single amino acid variant filtering, and synthesized peptide matching. Tissue expression analysis showed that 3 of 14 MPs were testis-specific proteins. Functional analysis showed that 10 of 14 MPs were closely related to liver tumor, liver carcinoma, and hepatocellular carcinoma. Another 100 MPs were listed as candidates but required additional verification information. All MS data sets have been deposited into the ProteomeXchange with the identifier PXD009737.

KEYWORDS:

Chromosome-Centric Human Proteome Project; missing proteins; multiprotease; reversed-phase HPLC; testis

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