Site-specific identification and quantitation of endogenous SUMO modifications under native conditions.
Site-specific identification and quantitation of endogenous SUMO modifications under native conditions.
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DOI：
10.1038/s41467-017-01271-3
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发表时间：
2017-10-27
影响因子：
16.6
通讯作者：
Komives EA
中科院分区：
文献类型：
--
作者：
Lumpkin RJ;Gu H;Zhu Y;Leonard M;Ahmad AS;Clauser KR;Meyer JG;Bennett EJ;Komives EA
小泛素样修饰物（SUMO）修饰调节许多细胞过程。与泛素不同，内源性SUMO化蛋白的检测受到SUMO蛋白c端尾部缺乏天然存在的蛋白酶位点的限制。在蛋白质组范围内检测目标蛋白上的sumo化位点通常需要将引入色氨酸位点的突变sumo异位表达。在这里，我们报告了一种使用α-裂解蛋白酶（WaLP）的蛋白质组范围、位点水平检测内源性SUMOylation的方法。在SUMO修饰位点，WaLP消化SUMO化蛋白产生含有SUMO残基二赖氨酸（KGG）的肽。利用先前开发的含kgg肽的免疫亲和分离以及质谱分析，我们鉴定了1209个独特的内源性SUMO修饰位点。我们还证明了蛋白酶体抑制对泛素和sumo修饰的蛋白质组的影响，使用了泛素化和sumo修饰的肽的平行定量。这种方法的进步使内源性SUMOylated蛋白在完全天然条件下的测定成为可能。SUMOylation是一种涉及多种生物学途径的翻译后修饰。在这里，作者描述了一种在本地条件下对SUMO附着位点进行全局分析的方法，该方法还允许并行确定SUMO和Ub附着。
Small ubiquitin-like modifier (SUMO) modification regulates numerous cellular processes. Unlike ubiquitin, detection of endogenous SUMOylated proteins is limited by the lack of naturally occurring protease sites in the C-terminal tail of SUMO proteins. Proteome-wide detection of SUMOylation sites on target proteins typically requires ectopic expression of mutant SUMOs with introduced tryptic sites. Here, we report a method for proteome-wide, site-level detection of endogenous SUMOylation that uses α-lytic protease, WaLP. WaLP digestion of SUMOylated proteins generates peptides containing SUMO-remnant diglycyl-lysine (KGG) at the site of SUMO modification. Using previously developed immuno-affinity isolation of KGG-containing peptides followed by mass spectrometry, we identified 1209 unique endogenous SUMO modification sites. We also demonstrate the impact of proteasome inhibition on ubiquitin and SUMO-modified proteomes using parallel quantitation of ubiquitylated and SUMOylated peptides. This methodological advancement enables determination of endogenous SUMOylated proteins under completely native conditions. SUMOylation is post-translational modification implicated in several biological pathways. Here the authors describe an approach for the global profiling of SUMO attachment sites under native conditions that also allows the parallel determination of SUMO and Ub attachments.