Enrichment of O-GlcNAc Modified Proteins by the Periodate Oxidation−Hydrazide Resin Capture Approach

Abstract

A chemical derivatization approach has been developed for the enrichment of O-GlcNAc modified proteins. The procedure is based on the isolation technique used for N-glycoproteins with appropriate modifications because of the differences in the two types of glycosylation: a prolonged periodate oxidation is followed by hydrazide resin capture, on-resin proteolytic digestion and release of the modified peptides by hydroxylamine. This enrichment strategy offers a fringe benefit in mass spectrometry analysis. Upon collisional activation the presence of the open carbohydrate ring leads to characteristic fragmentation facilitating both glycopeptide identification and site assignment. The enrichment protocol was applied to the Drosophila proteasome complex previously described as OGlcNAc modified. The O-GlcNAc modification was located on proteasome interacting proteins, deubiquitinating enzyme Faf (CG1945) and a ubiquitin-like domain containing protein (CG7546). Three other proteins were also found GlcNAc modified, a HSP70 homologue (CG2918), scribbled (CG5462) and the 205 kDa microtubule-associated protein (CG1483). Interestingly, in the HSP70 homologue the GlcNAc modification is attached to an asparagine residue of a N-glycosylation motif.

The roles ofO-linked β-N-acetylglucosamine in cardiovascular physiology and disease

“…One challenge to advancing the field lies in the modification itself, which is challenging to detect and resists genetic/pharmacological manipulation. There have been a number of recent advances in methodologies for detecting O-GlcNAc modified proteins (28, 75, 76, 81, 123, 141, 151, 158, 159, 163-166, 170, 171, 192, 193), mapping the sites of addition of OGlcNAc (11,14,81,141,151,170), probing the dynamics of O-GlcNAcylation (12,13,77,165,168,194), and genetic models in which O-GlcNAc levels can be manipulated (5,49,73,131,146,172). Together, this suite of technologies should allow researchers to answer some of the key remaining questions: 1) How is the specificity of OGT and O-GlcNAcase regulated, and how is this altered during aging or in disease models?…”

N- and O-Glycosylation in the Murine Synaptosome

“…The presence of such N-linked glycopeptides has been reported in O-GlcNAc studies following different enrichment strategies (40,56,57). Single GlcNAc molecules N-linked to asparagine are not cleaved efficiently by PNGase F, which would explain why they have not been detected in previous glycan-level analyses (58).…”

Development of a Novel Method for Analyzing Collagen O-glycosylations by Hydrazide Chemistry

“…There are several other ways to release glycopeptides from hydrazide resin. One is to convert the glycopeptides to oxime derivatives with aminooxy reagents on hydrazide resin (19). Although our hydrazide method enabled us to identify the O-glycosylation sites of collagen, the low oxidation efficiency and recovery rate of GHL/GGHL remain challenges for precise quantitative analysis.…”

“…Because galactose oxidase activity especially in the GGHL standard was markedly enhanced by the coordinated addition of catalase and HRP (23,24), we applied this three-enzyme system. We used acid/heat treatment to cleave the hydrazone bond so that the eluted peptides contained entire carbohydrate chains, which has also been previously achieved with alternative methods (18,19). Using the hydrazide method, we could identify the glycan structures concurrently with determining the position of Oglycosylated hydroxylysine.…”

“…Another study has analyzed sialylated glycopeptides with an intact glycan by cleaving the hydrazone bond using hydrochloric acid under cooling conditions (18). O-GlcNAc-modified glycopeptides were released from the resin using hydroxylamine thus converting the sugar to an oxime derivative (19). Although various approaches have been adopted, most applications of hydrazide chemistry have been restricted to analysis of the N-glycoproteome.…”