A Novel Quantitative Mass Spectrometry Platform for Determining Protein O-GlcNAcylation Dynamics

Abstract

Over the past decades, protein O-GlcNAcylation has been found to play a fundamental role in cell cycle control, metabolism, transcriptional regulation, and cellular signaling. Nevertheless, quantitative approaches to determine in vivo GlcNAc dynamics at a large-scale are still not readily available. Here, we have developed an approach to isotopically label O-GlcNAc modifications on proteins by producing 13 C-labeled UDP-GlcNAc from 13 C 6 -glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine protein O-GlcNAcylation turnover rates. First, an efficient enrichment method for O-GlcNAc peptides was developed with the use of phenylboronic acid solid-phase extraction and anhydrous DMSO. The near stoichiometry reaction between the diol of GlcNAc and boronic acid dramatically improved the enrichment efficiency. Additionally, our kinetic model for turnover rates integrates both metabolomic and proteomic data, which increase the accuracy of the turnover rate estimation. Other advantages of this metabolic labeling method include in vivo application, direct labeling of the O-GlcNAc sites and higher confidence for site identification. Concentrating only on nuclear localized GlcNAc modified proteins, we are able to identify 105 O-GlcNAc peptides on 42 proteins and determine turnover rates of 20 O-GlcNAc peptides from 14 proteins extracted from HeLa nuclei. In general, we found O-GlcNAcylation turnover rates are slower than those published for phosphorylation or acetylation. Nevertheless, the rates widely varied depending on both the protein and the residue modified. We believe this methodology can be broadly applied to reveal turnovers/dynamics of protein OGlcNAcylation from different biological states and will provide more information on the significance of O-GlcNAcylation, enabling us to study the temporal dynamics of this critical modification for the first time. Molecular &

Heavy Sugar and Heavy Water Create Tunable Intact Protein Mass Increases for Quantitative Mass Spectrometry in Any Feed and Organism

“…In the event that some amino acids are labeled at different rates than others, 33 and equilibrium labeling is not achieved, a bias similar to that observed with SILAC could be introduced to TIPMI. To address this potential caveat, the experimentally observed range of TIPMI peak shifts were compared to theoretical spacing of both TIPMI and SILAC (Figure 3).…”

Global and site‐specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry

“…Through this strategy, protein O‐GlcNAcylation turnover rates were determined. In total, they identified 105 O‐GlcNAcylated peptides from 42 proteins and determined the turnover rates of 20 O‐GlcNAcylated peptides from 14 proteins in the nuclei of HeLa cells (Wang et al, ). They found that although the rates widely varied depending on both the protein and the site, O‐GlcNAcylation turnover rates were generally slower than those published results for phosphorylation or acetylation.…”

Chemical Glycoproteomics

“…316 Most recently, ETD was used to quantify the dynamics in O-GlcNAcylation sites after metabolically labeling HeLa cells with 13 C 6 -glucose (which was converted to 13 C-labeled UDP-GlcNAc). 317 …”