Large-scale site-specific mapping of the O-GalNAc glycoproteome.

Nature Protocols
Weiming YangHui Zhang

Abstract

Protein glycosylation is one of the most common protein modifications. A major type of protein glycosylation is O-GalNAcylation, in which GalNAc-type glycans are attached to protein Ser or Thr residues via an O-linked glycosidic bond. O-GalNAcylation is thought to play roles in protein folding, stability, trafficking and protein interactions, and identification of the site-specific O-GalNAc glycoproteome is a crucial step toward understanding the biological significance of the modification. However, lack of suitable methodology, absence of consensus sequon of O-GalNAcylation sites and complex O-GalNAc glycan structures pose analytical challenges. We recently developed a mass spectrometry-based method called extraction of O-linked glycopeptides (EXoO) that enables large-scale mapping of site-specific mucin-type O-GalNAcylation sites. Here we provide a detailed protocol for EXoO, which includes seven stages of: (1) extraction and proteolytic digestion of proteins to peptides, (2) sequential guanidination and de-salting of peptides, (3) enrichment of glycopeptides, (4) solid-phase peptide conjugation and release of O-GalNAc glycopeptides using the OpeRATOR protease, (5) liquid chromatography with tandem mass spectrometry analysis ...Continue Reading

References

Aug 1, 1993·Molecular Microbiology·B Westerlund, T K Korhonen
Nov 20, 2003·Proteomics·Jun Ho Jang, Samir Hanash
Nov 24, 2004·The Journal of Immunology : Official Journal of the American Association of Immunologists·James N ArnoldPauline M Rudd
Jul 12, 2005·Bioorganic & Medicinal Chemistry·Howard C Hang, Carolyn R Bertozzi
Jun 8, 2007·Cancer Letters·Yelena V Kovtun, Victor S Goldmacher
Aug 4, 2007·Molecular & Cellular Proteomics : MCP·David M GoodJoshua J Coon
Oct 20, 2009·Nature Methods·Jonas NilssonGöran Larson
Nov 19, 2009·The FEBS Journal·Pia H JensenNicolle H Packer
Nov 26, 2009·Nature Reviews. Cancer·Donald W Kufe
Feb 11, 2010·Seminars in Cell & Developmental Biology·Lawrence A Tabak
Oct 11, 2011·Nature Methods·Catharina SteentoftHenrik Clausen
Mar 7, 2012·Molecular & Cellular Proteomics : MCP·Z DarulaK F Medzihradszky
Apr 16, 2013·The EMBO Journal·Catharina SteentoftHenrik Clausen
May 23, 2015·Molecular & Cellular Proteomics : MCP·Katalin F MedzihradszkyRobert J Chalkley
Nov 4, 2015·Nucleic Acids Research·Juan Antonio VizcaínoHenning Hermjakob
Nov 26, 2015·Molecular & Cellular Proteomics : MCP·Marcus HoffmannErdmann Rapp
Feb 15, 2017·Biochemistry·SriHariRaju MulagapatiT Shantha Raju
May 19, 2017·Nature Protocols·Shuang YangHui Zhang
Nov 11, 2017·Journal of Proteome Research·Jian-Ying ZhouHui Zhang
Dec 20, 2017·Journal of the American Society for Mass Spectrometry·Fränze MüllerJuri Rappsilber
Jun 26, 2018·Analytical Chemistry·Shuang YangJohn Cipollo
Nov 22, 2018·Molecular Systems Biology·Weiming YangHui Zhang
Mar 27, 2019·Proceedings of the National Academy of Sciences of the United States of America·Stacy A MalakerCarolyn R Bertozzi
Apr 9, 2019·Open Biology·Feifei ZhuKeping Chen
May 31, 2019·Minerva medica·Sarah M Moran, Daniel C Cattran

❮ Previous
Next ❯

Citations

Jan 14, 2021·Biochemical Society Transactions·Anastasia ChernykhMorten Thaysen-Andersen
Sep 18, 2020·Molecular & Cellular Proteomics : MCP·Nicholas M RileySharon J Pitteri
Feb 16, 2021·Molecular & Cellular Proteomics : MCP·Nicholas M RileySharon J Pitteri
Apr 29, 2021·The Biochemical Journal·D Judy ShonStacy A Malaker
Jul 3, 2021·Frontiers in Chemistry·Ding LiuLei Li
Aug 7, 2021·Current Opinion in Biotechnology·Yuanwei Xu, Hui Zhang
Aug 5, 2021·The FEBS Journal·Hans H WandallIeva Bagdonaite

❮ Previous
Next ❯

Methods Mentioned

BETA
electron transfer dissociation
GalNAcosylation
glycosylation
protein assay

Software Mentioned

ProteoWizard
Feature Mapper
MSConvertGUI
Proteome Discoverer
OpeRATOR
SEQUEST
DAVID
EXoO
SimpleCell
SEQUEST Excel

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.