• 研究论文 •

### 尺寸排阻-反相液相色谱-质谱联用技术在大鼠肾脏翻译后修饰蛋白质鉴定中的应用

1. 澳门科技大学中医药学院, 中药质量研究国家重点实验室, 澳门 999078
• 收稿日期:2020-05-29 出版日期:2021-01-08 发布日期:2020-12-20
• 通讯作者: 伍建林
• 作者简介:伍建林: 博士,现任澳门科技大学中药质量研究国家重点实验室副教授,博士生导师,广州医科大学南山学者特聘教授 (2017-2020)。2004年获日本新潟大学工学部工学硕士 (NMR应用),2009年获香港浸会大学化学系哲学博士 (MS应用),后在香港大学李嘉诚医学院从事博士后研究员工作并筹建了基于LC、MS和NMR的香港大学代谢组学与先端分析实验室,2011年加入澳门科技大学任助理教授,筹建了基于LC、MS和NMR的包括组学技术与创新药物研究中心和澳门核磁共振与光谱中心。目前主要从事多组学整合分析方法研究消化道肿瘤机制、药物和食品化学分析及应用等。截至目前,以第一和通讯作者 (含共同) 在Cell Res, Gastroenterology, Anal Chem, J Hazard Mater, Environ Int, Food Chem, J Agric Food ChemJ Nat Prod等,及合作在J Hepatol, Gastroenterology, Adv Sci等杂志上发表LC、MS和NMR应用相关SCI文章80余篇,授权国际发明专利5项。*Tel:(0853)88972406,E-mail:jlwu@must.edu.mo.
• 基金资助:
澳门特别行政区科学技术发展基金会(0090/2018/A3)

### Size exclusion-reverse liquid column chromatography-mass spectrometry and its application in the identification of post-translationally modified proteins in rat kidney

LI Jianmin, ZHUO Yue, ZHANG Yida, LI Na, WU Jianlin*()

1. Faculty of Chinese Medicine, State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau 999078, China
• Received:2020-05-29 Online:2021-01-08 Published:2020-12-20
• Contact: WU Jianlin
• Supported by:
the Science and Technology Development Fund, Macau SAR(0090/2018/A3)

LC-MS联用技术在蛋白质组学研究中具有重要的作用,但是在复杂的生物体系中,由于样品的高度复杂性和其中蛋白质含量的巨大差异,执行全面且无倾向的蛋白质组分析是一项挑战。因此,在液相色谱分离中采用基于不同原理的色谱分离方法来降低蛋白质样本的复杂度,并对微量蛋白质进行富集,对后续采用质谱方法进行信息的采集和深入分析至关重要。在这里我们开发了一种基于尺寸排阻色谱(SEC)与反相液相色谱(RPLC)结合的新方法来进行复杂体系蛋白质的分离和鉴定,特别是对于微量蛋白质的分析。首先使用SEC对蛋白质进行分离和富集,并酶解成多肽,再通过RPLC-MS联用的方法对酶解后的多肽进行分离和鉴定。结果显示使用上述方法可以有效降低蛋白质样本的复杂度,并有效提高微量蛋白质的鉴定能力,可从大鼠肾脏鉴定出23621个肽段及1345个蛋白质,比常规的二维强阳离子交换-反相液相色谱法(2D SCX-RPLC)鉴定到的肽段及蛋白质分别多出69%及27%。此外,该方法对肾脏翻译后修饰(PTM)蛋白质的鉴定显示出更多的优势,翻译后修饰的多肽鉴定率显著增加,特别是磷酸化肽段的鉴定效率可达到靶向富集策略的水平。在此展示的SEC-RPLC-MS可以更好地了解蛋白质翻译后修饰对肾脏的影响,最终将有助于增加我们对正常的生理性肾功能以及病理过程机制的理解。

Abstract:

Proteomics is an emerging field that has been shown to play a crucial role in unveiling the mechanisms underlying physiological and pathological processes, and liquid chromatography-mass spectrometry (LC-MS) is one of the most important methods employed in this field. However, in complex biological systems, such as eukaryotes, it is challenging to perform a comprehensive and unbiased proteome analysis due to the high complexity of biological samples and enormous differences in sample contents. For example, post-translational modifications (PTMs) in proteins are imperative for cell signaling, but post-translationally modified proteins account for about 1% of the total proteins in a single cell, making their identification extremely difficult. Therefore, chromatographic separation methods based on different principles are generally applied to reduce the complexity of biological samples and enrich trace proteins for their identification through mass spectrometry (MS). In this study, we developed a new proteomics method by combining size exclusion chromatography (SEC) and reversed-phase chromatography (RPLC), to separate and identify trace proteins in complex systems. SEC was used to separate and enrich kidney-specific proteins. After optimization of the method, it was found that 30 mmol/L of ammonium acetate could efficiently separate rat kidney proteins from the total protein fraction so that they could be eluted based on their relative molecular mass. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and LC-MS results showed that our SEC separation method not only refined the protein composition of the biological sample but also enhanced the relative contents of trace proteins through multiple injections. The collected protein fractions were further concentrated through ultrafiltration centrifugation followed by freeze-drying, which further improved the recovery of trace proteins by approximately 90% and largely decreased the time required with the use of freeze-drying alone. Thereafter, five protein fractions were separately digested using trypsin, and the resultant peptides were further analyzed by reverse phase chromatography-MS analysis. In the RPLC column, the peptides were isolated mainly based on their hydrophobicity. As a result, by combining SEC and RPLC, 23621 peptides and 1345 proteins were identified from the kidney, with an increase in numbers by 69% and 27%, respectively, when compared to those obtained using the common 2D strong cation exchange (SCX)-RPLC-MS method. However, no significant difference was observed in the pI and grand average of hydropathicity (GRAVY) values. Gene ontology (GO) analysis revealed an increase in the number of proteins in each cell component, especially the membrane. Furthermore, identification of a higher rate of identified peptides than proteins suggested that the protein coverage was also improved, thereby facilitating the detection of PTM proteins. Consequently, five common PTMs in biological processes, including methylation, acetylation, carbamylation, oxidation, and phosphorylation, were examined and compared between the two methods. As expected, the number of post-translationally modified peptides identified using SEC-RPLC-MS were 1.7-1.9 times more than those determined using the SCX-RPLC-MS method. Especially for the identification of phosphorylated peptides, we could achieve the level of the targeted enrichment strategy; however no significant difference was observed in the extents of phosphorylation among serine, threonine, and tyrosine. These results further indicate that upon combining SEC and RPLC, high efficiency could be achieved by decreasing the complexity of the protein sample, and the identification was unbiased. Finally, the phosphorylation of some kidney proteins, such as spectrin, L-lactate dehydrogenase, and ATPases, was found, which is critical for their functions. In summary, the SEC-RPLC-MS approach was developed for the identification of rat kidney proteins and is especially applicable for the identification of PTM proteins. Using this method, the identification efficiency for PTM peptides increased significantly. Therefore, this method has potential for better understanding the impact of PTM on kidney proteins and further elucidating the potential mechanisms underlying its physiological and pathological functions.