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)C6-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 O-GlcNAcylation 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.

Fig. 1.

A, Hexosamine biosynthetic pathway (HBP) and the metabolic labeling strategy of UDP-GlcNAc from ¹³C-glucose. Species that are heavy labeled are marked with an asterisk. B, Modification cycle of O-GlcNAcylation. Protein O-GlcNAcylation reaction involves a UDP-GlcNAc cofactor and two enzymes that are O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that add and remove the O-GlcNAc moiety, respectively. C, Workflow for the O-GlcNAc peptide analysis, including isotopic labeling, sample preparation, mass spectrometry, and quantification.

Fig. 2.

The production of ¹³C labeled UDP-GlcNAc metabolite from ¹³C₆-glucose and the reduction of normal light UDP-GlcNAc in HeLa cells over time. Relative abundances of unlabeled and labeled UDP-GlcNAc were quantified by using two different methods. Black and red traces were quantified from MS1 of all isotopic forms of UDP-GlcNAc. Blue and orange traces were quantified from MS/MS fragmentation ions of all isotopic forms of UDP-GlcNAc. Supporting supplemental Table S3 showed all values of the quantification. By fitting the relative abundances of heavily labeled UDP-GlcNAc over time to an exponential equation, a half-time rate of 8.9 h was determined for the production of 50% of GlcNAc labeled UDP-GlcNAc.

Fig. 3.

Fragmentation routes, product ions structures, and masses of the GlcNAc oxonium ion in HCD mass spectra of GlcNAc containing peptides. The heavily labeled oxonium ions have mass shifts compared with the light ones based on the numbers of carbon in the molecule.

Fig. 4.

Enrichment of an O-GlcNAc peptide standard spiked into a BSA peptide digest mixture. A, The formula and the structure of the reaction between 1,3-diol of GlcNAc and boronic acid. B, The comparison of the TIC between enriched O-GlcNAc peptide sample and input. C, Fold of enrichment of the GlcNAc peptide standard from the BSA peptide mixture. The fold enrichments were calculated and normalized by the ratio of the input. The elution contains both nonspecific binding peptides and the glycosylated peptide. We determined the ratio of total GlcNAc/BSA peptides of the input and the eluted GlcNAc/BSA peptides from the respective AUC of the extracted ion chromatogram. (Supporting supplemental Table S4).

Fig. 5.

A, Extracted ion chromatograms showed that light and heavy O-GlcNAc peptides from NUP153 with the sequence of FGVSSSSSGPSQTLTSTGNFK_1GlcNAc on Ser/Thr/Asn were eluted at the same retention time. The possible GlcNAc site is colored red in the sequence on top of panels. B, MS1 spectra for light and heavy labeled peptide containing GlcNAc. Heavy labeled peptides had mass shifts of 6.020 Da and 8.024 Da compared with light peptides, corresponding to the theoretic mass differences of 6.018 Da and 8.024 Da including 6 and 8 ¹³C atoms, respectively. The isotopic distribution of heavy peptides suggests that both species exist. C, Tandem mass spectra for the heavy in green and light in red labeled peptides with GlcNAc modification. Fragment ions with the GlcNAc loss have the same mass as the corresponding light one. Depending on the position of the GlcNAc, the b and y ions might also be b/y ions with glcnac loss. Therefore, we label them as b′ or y′ representing either b/y ion peaks or b/y with glcnac neutral loss ion peaks. An 8-Da shift is present in GlcNAc containing ions such as [M+2H]²⁺⁺ and GlcNAc oxonium product ion.

Fig. 6.

Increase in relative abundance of heavy labeled O-GlcNAc peptides over time. A, Paired light peptide (black) and heavy peptides (red) eluted at similar retention times across all time points with a decrease of the relative abundance of light peptide and an increase of the relative abundance of the heavy peptide. B, Progress curve demonstrating the increased relative abundance of a heavy peptide with the sequence and modification of FGVSSSSSGPSQTLTSTGNFK_1GlcNAc on S/T/N over time. Error bars are obtained from two technical replicates.

Fig. 7.

Cartoon illustration for the labeling process. U represents unlabeled unmodified peptide and G* represents labeled O-GlcNAc peptide. AA represents unlabeled amino acid. Glucose* represents labeled glucose. AA* represents labeled amino acids. U* represents labeled unmodified proteins. F*_OGT is the O-GlcNAcylation flux of a labeled O-GlcNAc peptide catalyzed by OGT. F*_OGA is the de-GlcNAcylation flux of a labeled O-GlcNAc peptide catalyzed by OGA. F*_Deg is the degradation flux of a labeled O-GlcNAc peptide through protein synthesis. F*_HBP is the flux of hexosamine biosynthetic pathway to form labeled UDP-GlcNAc from labeled glucose. F*_AA is the flux of labeled amino acid synthesis from labeled glucose. F*_U is the flux of labeled unmodified protein synthesis from labeled amino acids. In our model as shown in Eq 2, the variables inside the frame were considered to determine protein O-GlcNAcylation turnover rates. Relative abundances of UDP-GlcNAc* and relative abundances U (colored blue) at a specific time were calculated independently. Relative abundances of G* (colored red) were measured at experimental time points in the glucose labeling experiment.

Fig. 8.

Progress curves showing increased relative abundance of heavy O-GlcNAc labeled peptides on Host Cell Factor 1 over time. Sequences and sites of modification of peptides are SPITIITT801_GlcNAcK; VT490_GlcNAcGPQATTGTPLVTM_oxRPASQAGK; T579_GlcNAcM_oxAVTPGTTTLPATVK; Turnover rates (k₁ and k_-1) of different O-GlcNAc peptides were determined. Error bars are obtained from two technical replicates.