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

Abstract

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.

Systematic analysis of the impact of phosphorylation and O-GlcNAcylation on protein subcellular localization

“…As phosphorylated and O-GlcNAcylated proteins are typically of low abundances in cells, different enrichment methods were used for their global analysis by liquid chromatography-mass spectrometry (LC-MS). 9 – 13 In this study, metabolic labeling with Ac 4 GalNAz was used to label O-GlcNAcylated proteins for their enrichment, 14 – 16 and phosphorylated peptides were enriched using the TiO 2- based method. 17 , 18 O-GlcNAcylated, phosphorylated, and non-modified peptides were labeled with the TMT reagents for quantification of the nuclear-cytoplasmic distributions of proteins.…”

Standards-Free Absolute Quantitation of Oxidizable Glycopeptides by Coulometric Mass Spectrometry

“…Recent advances in instrumentation, , sample preparation techniques − and enrichment strategies − have fortified MS-based glycoproteomics in more advanced applications. The most common approach for quantifying glycans or glycopeptides is based on relative quantitation.…”

Deciphering the Properties and Functions of Glycoproteins Using Quantitative Proteomics

“…19 It was recently unveiled that this modification can occur cotranslationally, and it can modify many proteins in human cells. 20,21 When a protein is glycosylated, its hydrophobicity, structure, dynamics, and interactions with other molecules can be dramatically changed. Despite the importance of protein glycosylation, it is still very challenging to globally and sitespecifically analyze glycoproteins due to the following reasons.…”

“…For this modification, O- GlcNAc is attached to the serine and threonine residues catalyzed by the sole enzyme of O- GlcNAc transferase (OGT), and is only removed by O- GlcNAcase (OGA). − This modification is highly dynamic and is involved in crosstalk with other modifications, including phosphorylation . It was recently unveiled that this modification can occur cotranslationally, and it can modify many proteins in human cells. , …”

“… 19 It was recently unveiled that this modification can occur cotranslationally, and it can modify many proteins in human cells. 20 , 21 …”