Methods for Enrichment and Assignment of N-Acetylglucosamine Modification Sites

O-GlcNAcylation, the addition of a single N-acetylglucosamine residue to serine and threonine residues of cytoplasmic, nuclear, or mitochondrial proteins, is a widespread regulatory posttranslational modification. It is involved in the response to nutritional status and stress, and its dysregulation is associated with diseases ranging from Alzheimer’s to diabetes. Although the modification was first detected over 35 years ago, research into the function of O-GlcNAcylation has accelerated dramatically in the last 10 years owing to the development of new enrichment and mass spectrometry techniques that facilitate its analysis. This article summarizes methods for O-GlcNAc enrichment, key mass spectrometry instrumentation advancements, particularly those that allow modification site localization, and software tools that allow analysis of data from O-GlcNAc-modified peptides. [Display omitted] •O-GlcNAc enrichment methods•Mass spectrometry methods for O-GlcNAc analysis and site identification•Software tools to assist in O-GlcNAc data analysis•New dataset identifies over 700 sites of O-GlcNAc modification in human monocytes This review article summarizes methods for O-GlcNAc enrichment and different mass spectrometric approaches for acquiring data on modified peptides and describes software strategies for analyzing data, including the challenges of reliably identifying modification sites and differentiating between other potential HexNAc modifications. It then presents a new dataset to exemplify what is currently achievable.