Novel Antibodies for the Simple and Efficient Enrichment of Native O-GlcNAc Modified Peptides

Autor: Stacy A. Malaker, Samuel A. Myers, Xiang Li, Anjana Rao, Steven A. Carr, Johain R. Ounadjela, Borislav Dejanovic, Rajan A. Burt, Hayley J. Peckham, Kimberly Lee
Jazyk: angličtina
Rok vydání: 2021
Předmět:
Glycan
enrichment
SA
sialic acid
Immunoprecipitation
medicine.drug_class
CID
collision-induced dissociation
Lysine
WGA
wheat germ agglutinin
pd
product-dependent
Monoclonal antibody
N-Acetylglucosaminyltransferases
Biochemistry
HCD product-dependent ETD
Analytical Chemistry
CE
collision energy
XICs
extracted ion chromatograms
oxonium ion ratios
ETD
electron transfer dissociation
Mice
Ubiquitin
GalNAc
N-acetylgalactosamine
mESCs
mouse embryonic stem cells
HexHexNAc
Hexose-N-acetylhexosamine
medicine
glycoproteomics
Animals
bRP
basic reversed-phase
pd-EThcD
product-dependent triggered EThcD
Molecular Biology
biology
Chemistry
LC-MS/MS
liquid chromatography–tandem mass spectrometry
Technological Innovation and Resources
Glycopeptides
Antibodies
Monoclonal
Brain
Mouse Embryonic Stem Cells
GlcNAc
N-acetylglucosamine
Glycoproteomics
Electron-transfer dissociation
carbohydrates (lipids)
Acetylation
HexNAc
N-acetylhexosamine
HCD
higher-energy collisional dissociation
biology.protein
O-GlcNAc
EThcD
electron transfer dissociation with supplemental HCD activation
Abs
antibodies
Zdroj: Molecular & Cellular Proteomics : MCP
ISSN: 1535-9484
1535-9476
Popis: Antibodies against posttranslational modifications (PTMs) such as lysine acetylation, ubiquitin remnants, or phosphotyrosine have resulted in significant advances in our understanding of the fundamental roles of these PTMs in biology. However, the roles of a number of PTMs remain largely unexplored due to the lack of robust enrichment reagents. The addition of N-acetylglucosamine to serine and threonine residues (O-GlcNAc) by the O-GlcNAc transferase (OGT) is a PTM implicated in numerous biological processes and disease states but with limited techniques for its study. Here, we evaluate a new mixture of anti-O-GlcNAc monoclonal antibodies for the immunoprecipitation of native O-GlcNAcylated peptides from cells and tissues. The anti-O-GlcNAc antibodies display good sensitivity and high specificity toward O-GlcNAc-modified peptides and do not recognize O-GalNAc or GlcNAc in extended glycans. Applying this antibody-based enrichment strategy to synaptosomes from mouse brain tissue samples, we identified over 1300 unique O-GlcNAc-modified peptides and over 1000 sites using just a fraction of sample preparation and instrument time required in other landmark investigations of O-GlcNAcylation. Our rapid and robust method greatly simplifies the analysis of O-GlcNAc signaling and will help to elucidate the role of this challenging PTM in health and disease.
Graphical Abstract
Highlights • Anti-O-GlcNAc antibodies are fast and simple enrichment reagents. • Anti-O-GlcNAc antibodies are sensitive and achieve significant depth of coverage. • Anti-O-GlcNAc antibodies are specific for singular O-GlcNAc modifications. • Anti-O-GlcNAc antibody enrichment techniques can be applied to cells and tissues. • HCD product-triggered EThcD data acquisition improves depth of coverage.
In Brief O-GlcNAc, the single N-acetylglucosamine coupled to serine and threonines of nucleocytoplasmic proteins, is an enigmatic posttranslational modification implicated in most cellular functions. Methods to study the O-GlcNAc modified proteome have been challenging, in part, due to limitations in its enrichment from native tissue. Here, we characterize novel antibodies for the enrichment of native O-GlcNAc modified peptides. Our enrichment strategy shows strong sensitivity and excellent specificity for O-GlcNAc. We expect these reagents to be a significant advancement for the field.
Databáze: OpenAIRE
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