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J Proteome Res. 2018 Dec 7;17(12):4152-4159. doi: 10.1021/acs.jproteome.8b00409. Epub 2018 Oct 26.

Improvement of Peptide Separation for Exploring the Missing Proteins Localized on Membranes.

Zhang Y1,2, Lin Z1,2, Hao P3, Hou K4, Sui Y4, Zhang K1,2, He Y1,2, Li H5, Yang H1,6, Liu S1,2, Ren Y1,2.

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BGI-Shenzhen , Beishan Industrial Zone 11th building , Yantian District, Shenzhen , Guangdong 518083 , China.
China National GeneBank , BGI-Shenzhen , Jinsha Road , Shenzhen 518120 , China.
School of Life Science and Technology , ShanghaiTech Universit y, 393 Middle Huaxia Road , Shanghai 201210 , China.
The Second Maternal and Child Health Care Center of Huangdao District , 236 Fuchunjiang Road , Qingdao 266555 , Shandong , China.
Pulmonary Function Room , Shenzhen Seventh People's Hospital , 2010 Wutong Road , Yantian District, Shenzhen , Guangdong 518081 , China.
James D. Watson Institute of Genome Sciences , Hangzhou 310058 , China.


Following an enormous effort by the global scientific community coordinated by HUPO's Human Proteome Project, the number of proteins without high-quality MS or other evidence (colloquially termed missing proteins) has substantially decreased; however, some highly hydrophobic MPs remain on the list. We believe that efficient peptide separation is an approach that can be used to improve the identification of these hydrophobic MPs. We propose that peptides prepared from the membrane fractions of human cell lines and placental tissue can be well separated from hydrophilic peptides in organic solvents at high concentrations due to the precipitation of hydrophilic peptides with lower solubility. Using a combination strategy of peptide separation in 98% acetonitrile prior to traditional 2D reverse-phase liquid chromatography, more hydrophobic peptides were detected in the supernatants of the organic solvent extractions than were found in the pellets. When this strategy was adopted, 30 MPs (≥2 non-nested unique peptides with ≥9 amino acids) with 114 unique peptides were identified at protein false discovery rate (FDR) < 1%, including 7, 12, and 13 MPs obtained from membrane preparations derived from K562, HeLa cells, and human placenta, respectively. Of the 30 MPs identified in this study, 19 were categorized as membrane proteins or extracellular matrix proteins. Furthermore, 20 were verified to possess two non-nested unique peptides through parallel reaction monitoring with the corresponding chemically synthesized peptides. The use of organic solvents at high concentrations was shown to be an efficient way to improve the exploration of hydrophobic MPs. The data obtained in this study are available via ProteomeXchange (PXD010630) and PeptideAtlas (PASS01218).


LC−MS/MS; high concentration organic solvents; membrane fraction; missing proteins

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