Combining Selective Enrichment and a Boosting Approach to Globally and Site-Specifically Characterize Protein Co-translational O‑GlcNAcylation

Protein O-GlcNAcylation plays extremely
important
roles in mammalian cells, regulating signal transduction and gene
expression. This modification can happen during protein translation,
and systematic and site-specific analysis of protein co-translational O-GlcNAcylation can advance our understanding of this important
modification. However, it is extraordinarily challenging because normally O-GlcNAcylated proteins are very low abundant and the abundances
of co-translational ones are even much lower. Here, we developed a
method integrating selective enrichment, a boosting approach, and
multiplexed proteomics to globally and site-specifically characterize
protein co-translational O-GlcNAcylation. The boosting
approach using the TMT labeling dramatically enhances the detection
of co-translational glycopeptides with low abundance when enriched O-GlcNAcylated peptides from cells with a much longer labeling
time was used as a boosting sample. More than 180 co-translational O-GlcNAcylated proteins were site-specifically identified.
Further analyses revealed that among co-translational glycoproteins,
those related to DNA binding and transcription are highly overrepresented
using the total identified O-GlcNAcylated proteins
in the same cells as the background. Compared with the glycosylation
sites on all glycoproteins, co-translational sites have different
local structures and adjacent amino acid residues. Overall, an integrative
method was developed to identify protein co-translational O-GlcNAcylation, which is very useful to advance our understanding
of this important modification.

蛋白质O-葡萄糖基化在哺乳动物细胞中发挥着至关重要的角色，调节信号转导和基因表达。该修饰可发生在蛋白质翻译过程中，对蛋白质共翻译O-葡萄糖基化的系统性和位点特异性分析，能够深化我们对这一重要修饰机制的理解。然而，这一过程极具挑战性，因为通常O-葡萄糖基化蛋白质含量极低，而共翻译形式的含量则更为稀少。在本研究中，我们开发了一种整合选择性富集、增强方法和多重质谱分析的方法，以实现对蛋白质共翻译O-葡萄糖基化的全局和位点特异性表征。利用TMT标记的增强方法显著提高了检测低丰度共翻译糖肽的能力，当使用具有更长标记时间的细胞中富集的O-葡萄糖基化肽作为增强样本时尤为显著。我们成功鉴定了超过180种共翻译O-葡萄糖基化蛋白质。进一步分析显示，在共翻译糖蛋白中，与DNA结合和转录相关的蛋白质在总鉴定出的O-葡萄糖基化蛋白质中高度富集。与所有糖蛋白上的糖基化位点相比，共翻译位点具有不同的局部结构和邻近氨基酸残基。总体而言，一种综合方法被开发出来以识别蛋白质共翻译O-葡萄糖基化，这对于深化我们对这一重要修饰机制的理解极为有用。