Improvement of Peptide Separation for Exploring the Missing Proteins Localized on Membranes,Journal of Proteome Research

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Improvement of Peptide Separation for Exploring the Missing Proteins Localized on Membranes
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2018-10-26 , DOI: 10.1021/acs.jproteome.8b00409
Yuanliang Zhang _{1,

2} , Zhilong Lin _{1,

2} , Piliang Hao ₃ , Kexia Hou ₄ , Yuanyuan Sui ₄ , Keren Zhang _{1,

2} , Yanbin He _{1,

2} , Hong Li ₅ , Huanming Yang _{1,

6} , Siqi Liu _{1,

2} , Yan Ren _{1,

2}

Affiliation

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).

中文翻译：

改进用于分离膜上缺失蛋白的肽分离方法

在HUPO人类蛋白质组计划的协调下，全球科学界做出了巨大努力，没有高质量MS或其他证据的蛋白质（俗称缺失蛋白）的数量大大减少了；但是，一些高度疏水的MP仍在列表中。我们认为有效的肽分离是一种可用于改善对这些疏水性MP的鉴定的方法。我们建议，由人细胞系和胎盘组织的膜级分制备的肽可以在高浓度下与亲水性肽良好地分离，这是由于具有较低溶解度的亲水性肽的沉淀所致。在传统的2D反相液相色谱分析之前，采用在98％乙腈中分离肽的组合策略，与沉淀相比，在有机溶剂提取物的上清液中检测到的疏水肽更多。当采用此策略时，以蛋白质错误发现率（FDR）<1％鉴定出具有114个独特肽的30 MP（≥2个具有≥9个氨基酸的非嵌套独特肽），包括从以下方法获得的7 MP，12 MP和13 MP分别来自K562，HeLa细胞和人胎盘的膜制剂。在这项研究中鉴定出的30个MP中，有19个被分类为膜蛋白或细胞外基质蛋白。此外，通过与相应的化学合成肽的平行反应监测，证实了20个具有两个非嵌套的独特肽。高浓度的有机溶剂的使用被证明是改进对疏水性MP的探索的有效方法。

更新日期：2018-10-27

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