Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093-0378, USA.
Proteomic Service Group Cell Signaling Technology, 3 Trask Lane, Danvers, MA, 01923, USA.
Nat Commun. 2017 Oct 27;8(1):1171. doi: 10.1038/s41467-017-01271-3.
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.
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 末端尾部缺乏天然存在的蛋白酶位点的限制。靶蛋白上 SUMO 化位点的全蛋白质组检测通常需要表达带有引入的胰蛋白酶位点的突变 SUMO。在这里，我们报告了一种使用α-溶菌酶（WaLP）进行全蛋白质组、位点水平检测内源性 SUMO 化的方法。WaLP 消化 SUMO 化蛋白会在 SUMO 修饰位点生成含有 SUMO-残基二甘酰-赖氨酸（KGG）的肽。使用先前开发的含有 KGG 的肽的免疫亲和分离，然后进行质谱分析，我们鉴定了 1209 个独特的内源性 SUMO 修饰位点。我们还通过平行定量鉴定泛素化和 SUMO 化肽，证明了蛋白酶体抑制对泛素化和 SUMO 化蛋白质组的影响。这种方法学上的进步使在完全天然条件下确定内源性 SUMO 化蛋白成为可能。
