The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.

泛素结合酶 UBE2W 催化蛋白质 N 端的非规范泛素化，尽管其底物谱仍不清楚。为了鉴定内源性 N 端泛素化底物，我们发现了四种单克隆抗体，它们选择性地识别带有 N 端双甘氨酸残基的胰蛋白酶肽，对应于 N 端泛素化位点。重要的是，这些抗体不能识别赖氨酸上与异肽连接的双甘氨酸（泛素）修饰。我们解析了一种与 Gly-Gly-Met 肽结合的此类抗体的结构，以揭示其选择性识别的分子基础。我们将这些抗体与质谱蛋白质组学结合使用，以在 UBE2W 的内源性底物上绘制 N 端泛素化位点。这些底物包括 UCHL1 和 UCHL5，其中 N 端泛素化明显改变了去泛素化酶 (DUB) 的活性。这项工作描述了一种抗体工具包，用于富集和全局分析内源性 N 端泛素化位点，同时揭示 UBE2W 的功能相关底物。