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J Proteome Res. 2018 Dec 7;17(12):4178-4185. doi: 10.1021/acs.jproteome.8b00398. Epub 2018 Oct 16.

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

He C1,2, Sun J2,3, Shi J2,3, Wang Y2, Zhao J2,4, Wu S2,4, Chang L2, Gao H2, Liu F3, Lv Z3, He F2, Zhang Y1,2, Xu P2,3,4.

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State Key Laboratory of Biocontrol, Guangdong Key Laboratory of Plant Resources, School of Life Sciences , Sun Yat-sen University , Guangzhou 510275 , China.
State Key Laboratory of Proteomics, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics , Beijing Proteome Research Center , Beijing 102206 , China.
Hebei Province Key Lab of Research and Application on Microbial Diversity, College of Life Sciences , Hebei University , Baoding , Hebei 071002 , China.
Key Laboratory of Combinational Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, School of Pharmaceutical Science , Wuhan University , Wuhan 430072 , China.


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 low-molecular-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 MS2 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 gel-elution 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.


Chromosome-centric Human Proteome Project; low molecular weight; missing proteins; testis; “mirror protease”

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