BGI-Shenzhen , Beishan Industrial Zone 11th building , Yantian District, Shenzhen , Guangdong 518083 , China.
Clinical laboratory of BGI Health , BGI-Shenzhen , Shenzhen 518083 , China.
J Proteome Res. 2019 Dec 6;18(12):4180-4188. doi: 10.1021/acs.jproteome.9b00353. Epub 2019 Nov 8.
Identifying more missing proteins (MPs) is an important mission of C-HPP. With the number of identified MPs being attenuated year by year (2,949 to 2,129 MPs from 2016 to 2019), we have realized that the difficulty of exploring the remaining MPs is a challenge in technique. Herein, we propose a comprehensive strategy to effectively enrich, separate, and identify proteins with low molecular weights, aiming at the discovery of MPs. Basically, a protein extract from human placenta was passed through a C18 SPE column, and the bound proteins that were eluted were further separated with an SDS-PAGE gel or a 50 kDa cutoff filter. The separated proteins were subjected to trypsin digestion, and the MS/MS signals were searched against data sets with two different digestion modes (full-trypsin and semitrypsin). The strategy was adopted, resulting in the identification of 4 MPs with 8 unique peptides (≥2 non-nested unique peptides with ≥9 amino acids). Importantly, the identification of 6 out of 8 of the unique peptides derived from the MPs was further supported by parallel reaction monitoring, which confirmed the identification of 3 MPs from human placenta tissues (Q6NT89: TMF-regulated nuclear protein 1; A0A183: late cornified envelope protein 6A; and Q6UWQ7: insulin growth factor-like family member 2, mapped to chromosomes 1, 1, and 19, respectively). The three proteins ranged in length from 80 aa to 227 aa. The study not only establishes a feasible strategy for analyzing proteins with low molecular weights but also fills a small part of a large gap in the list of MPs. The data obtained in this study are available via ProteomeXchange (PXD014083) and PeptideAtlas (PASS01389).
鉴定更多的缺失蛋白 (MPs) 是 C-HPP 的重要任务。随着鉴定的 MPs 数量逐年减少（2016 年至 2019 年从 2949 个减少到 2129 个 MPs），我们已经意识到探索剩余 MPs 的难度是技术上的一个挑战。在此，我们提出了一种综合策略，以有效地富集、分离和鉴定低分子量的蛋白质，旨在发现 MPs。基本上，从人胎盘提取的蛋白质提取物通过 C18 SPE 柱，洗脱结合的蛋白质，然后用 SDS-PAGE 凝胶或 50 kDa 截止过滤器进一步分离。分离的蛋白质进行胰蛋白酶消化，将 MS/MS 信号与两种不同消化模式（全胰蛋白酶和半胰蛋白酶）的数据进行搜索。采用该策略，共鉴定到 4 个 MPs，有 8 个独特肽（≥2 个非嵌套独特肽，每个肽≥9 个氨基酸）。重要的是，来自 MPs 的 8 个独特肽中的 6 个的鉴定结果得到平行反应监测的进一步支持，这证实了 3 个 MPs 从人胎盘组织中得到鉴定（Q6NT89：TMF 调节核蛋白 1；A0A183：晚期角蛋白包膜蛋白 6A；Q6UWQ7：胰岛素样生长因子家族成员 2，分别定位在 1、1 和 19 号染色体上）。这三种蛋白质的长度从 80 个氨基酸到 227 个氨基酸不等。该研究不仅建立了一种分析低分子量蛋白质的可行策略，而且填补了 MPs 列表中一小部分的空白。本研究获得的数据可通过 ProteomeXchange（PXD014083）和 PeptideAtlas（PASS01389）获取。
