A Proteomics Strategy for the Identification of FAT10-Modified Sites by Mass Spectrometry

The ubiquitin-like protein FAT10 (HLA-F adjacent transcript 10) is uniquely expressed in mammals. The fat10 gene is encoded in the MHC class I locus in the human genome and is related to some specific processes, such as apoptosis, immune response, and cancer. However, biological knowledge of FAT10 is limited, owing to the lack of identification of its conjugates. FAT10 covalently modifies proteins in eukaryotes, but only a few substrates of FAT10 have been reported until now, and no FATylated sites have been identified. Here, we report the proteome-scale identification of FATylated proteins by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We identified 175 proteins with high confidence as FATylated candidates. A total of 13 modified sites were identified for the first time by a modified search of the raw MS data. The modified sites were highly enriched with hydrophilic amino acids. Furthermore, the FATylation processes of hnRNP C2, PCNA, and PDIA3 were verified by a coimmunoprecipitation assay. We confirmed that most of the substrates were covalently attached to a FAT10 monomer. The functional distribution of the FAT10 targets suggests that FAT10 participates in various biological processes, such as translation, protein folding, RNA processing, and macromolecular complex assembly. These results should be very useful for investigating the biological functions of FAT10.泛素样蛋白FAT10（ubiquitin-like protein FAT10，亦称HLA-F相邻转录本10）在哺乳动物中呈特异性表达。FAT10基因定位于人类基因组的主要组织相容性复合体I类基因座（MHC class I locus），与细胞凋亡、免疫应答及癌症等特定生物学过程相关。但由于缺乏对其共价结合底物的鉴定手段，目前学界对FAT10的生物学认知仍十分有限。FAT10可在真核生物中对蛋白质进行共价修饰，但截至目前仅报道了少量FAT10修饰底物，且尚未有FAT10修饰位点被成功鉴定。本研究借助液相色谱-串联质谱法（LC-MS/MS），完成了蛋白质组规模的FAT10修饰蛋白鉴定工作。我们以高置信度鉴定得到175个FAT10修饰候选蛋白，并通过对原始质谱数据进行修饰位点搜索，首次鉴定出13个修饰位点。这些修饰位点显著富集于亲水性氨基酸残基。此外，我们通过免疫共沉淀实验（coimmunoprecipitation assay）验证了异质性细胞核核糖蛋白C2（hnRNP C2）、增殖细胞核抗原（PCNA）及蛋白质二硫键异构酶A3（PDIA3）的FAT10修饰过程，并证实大多数底物可与FAT10单体形成共价结合。对FAT10靶标的功能分布分析显示，FAT10参与了翻译、蛋白质折叠、RNA加工及大分子复合物组装等多种生物学过程。本研究成果将为后续FAT10生物学功能的相关研究提供重要参考。