From the ‡Chair of Proteomics and Bioanalytics, Technical University of Munich, Freising, Germany.
§Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan.
Mol Cell Proteomics. 2018 Jul;17(7):1378-1391. doi: 10.1074/mcp.RA118.000696. Epub 2018 Apr 2.
Citrullination is a posttranslational modification of arginine catalyzed by five peptidylarginine deiminases (PADs) in humans. The loss of a positive charge may cause structural or functional alterations, and while the modification has been linked to several diseases, including rheumatoid arthritis (RA) and cancer, its physiological or pathophysiological roles remain largely unclear. In part, this is owing to limitations in available methodology to robustly enrich, detect, and localize the modification. As a result, only a few citrullination sites have been identified on human proteins with high confidence. In this study, we mined data from mass-spectrometry-based deep proteomic profiling of 30 human tissues to identify citrullination sites on endogenous proteins. Database searching of ∼70 million tandem mass spectra yielded ∼13,000 candidate spectra, which were further triaged by spectrum quality metrics and the detection of the specific neutral loss of isocyanic acid from citrullinated peptides to reduce false positives. Because citrullination is easily confused with deamidation, we synthetized ∼2,200 citrullinated and 1,300 deamidated peptides to build a library of reference spectra. This led to the validation of 375 citrullination sites on 209 human proteins. Further analysis showed that >80% of the identified modifications sites were new, and for 56% of the proteins, citrullination was detected for the first time. Sequence motif analysis revealed a strong preference for Asp and Gly, residues around the citrullination site. Interestingly, while the modification was detected in 26 human tissues with the highest levels found in the brain and lung, citrullination levels did not correlate well with protein expression of the PAD enzymes. Even though the current work represents the largest survey of protein citrullination to date, the modification was mostly detected on high abundant proteins, arguing that the development of specific enrichment methods would be required in order to study the full extent of cellular protein citrullination.
精氨酸的瓜氨酸化是一种翻译后修饰，由人类的 5 种肽基精氨酸脱亚氨酶（PAD）催化。正电荷的丧失可能导致结构或功能的改变，虽然这种修饰与几种疾病有关，包括类风湿关节炎（RA）和癌症，但它的生理或病理生理作用在很大程度上仍不清楚。部分原因是由于现有方法学的局限性，无法有效地富集、检测和定位这种修饰。因此，只有少数精氨酸瓜氨酸化位点在人类蛋白质上被高可信度地鉴定出来。在这项研究中，我们从基于质谱的 30 个人体组织的深度蛋白质组学分析数据中挖掘，以鉴定内源性蛋白质上的瓜氨酸化位点。对约 7000 万个串联质谱的数据库搜索产生了约 13000 个候选谱，进一步通过谱质量指标和从瓜氨酸化肽中检测异氰酸的特定中性丢失来进行分类，以减少假阳性。由于瓜氨酸化很容易与脱酰胺混淆，我们合成了约 2200 个瓜氨酸化和 1300 个脱酰胺肽，以构建参考谱库。这导致在 209 个人类蛋白质上验证了 375 个瓜氨酸化位点。进一步的分析表明，>80%的鉴定修饰位点是新的，对于 56%的蛋白质，首次检测到瓜氨酸化。序列基序分析显示出对 Asp 和 Gly 的强烈偏好，这两种残基位于瓜氨酸化位点周围。有趣的是，虽然该修饰在 26 个人体组织中均有检测到，其中大脑和肺中的含量最高，但瓜氨酸化水平与 PAD 酶的蛋白质表达相关性不大。尽管目前的工作是迄今为止对蛋白质瓜氨酸化的最大调查，但该修饰主要在高丰度蛋白上检测到，这表明需要开发特定的富集方法，以便研究细胞蛋白质瓜氨酸化的全部程度。
