Optimization of Chemoenzymatic Mass Tagging by Strain-Promoted Cycloaddition (SPAAC) for the Determination of O-GlcNAc Stoichiometry by Western Blotting

Abstract

The dynamic modification of intracellular proteins by O-linked β -N-acetylglucosamine (O-GlcNAcylation) plays critical roles in many cellular processes. Although various methods have been developed for O-GlcNAc detection, there are few techniques for monitoring glycosylation stoichiometry and state (i.e., mono-, di-, etc., O-GlcNAcylated). Measuring the levels of O-GlcNAcylation on a given substrate protein is important for understanding the biology of this critical modification and for prioritizing substrates for functional studies. One powerful solution to this limitation involves the chemoenzymatic installation of polyethylene glycol polymers of defined molecular mass onto O-GlcNAcylated proteins. These "mass tags" produce shifts in protein migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that can be detected by Western blotting. Broad adoption of this method by the scientific community has been limited, however, by a lack of commercially available reagents and well-defined protein standards. Here, we develop a "click chemistry" approach to this method using entirely commercial reagents and confirm the accuracy of the approach using a semisynthetic O-GlcNAcylated protein. Our studies establish a new, expedited experimental workflow and standardized methods that can be readily utilized by non-experts to quantify the O-GlcNAc stoichiometry and state on endogenous proteins in any cell or tissue lysate.

MYPT1 O-GlcNAc modification controls the sensitivity of fibroblasts to sphingosine-1-phosphate mediated cellular contraction

“…However, the most obvious change was an overall decrease in our ability to detect MYTP1. We believe that this is due to the reduced affinity of some antibodies to highly PEGylated proteins, which we previously found for Nup62 49 . In an attempt to overcome this issue, we next generated NIH3T3 cells that stably express a FLAG-tagged version of MYPT1 (Supplementary Figure 5) and performed the same mass-shifting analysis with an anti-FLAG antibody (Figure 5f).…”

“…To accomplish this, we again took advantage of the chemoenzymatic methodology (Figure 5a), but instead of installing an enrichment tag, we modified each O-GlcNAc moiety with a polyethylene glycol (PEG) chain of either 2 or 5 kDa in molecular weight. This mass-shifting approach causes the O-GlcNAcylated fraction of a protein to run higher on an SDS-PAGE gel for subsequent analysis by Western blotting 48,49 . We applied this technique to NIH3T3 cells under three sets of conditions: 5SGlcNAc treatment (200 μM) to lower O-GlcNAcylation levels, DMSO vehicle to leave them unchanged, or Thiamet-G treatment (10 μM) to raise them.…”

Exploring structural dynamics of a membrane protein by combining bioorthogonal chemistry and cysteine mutagenesis

“…Although the pull-down observed with the azide-reactive DBCO-sulfo-biotin is comparable to that with the amine-reactive NHS-sulfo-biotin (Figure 1E, lane 2 and 4 and Figure 1G), suggesting that the extent of AHA incorporation is robust, this assay is relatively qualitative. We are currently developing quantitative methods to measure the extent of AHA incorporation using mass spectrometry and/or azide-reactive cyclooctyne polyethylene glycol (PEG) polymers (Darabedian et al, 2018). Background labeling of cysteine and methionine residues present within endogenously expressed proteins might also prevent detection of distinct fluorescent signals from proteins that do not express to high enough levels.…”

The O-GlcNAc Modification on Kinases

“…If the glycoprotein is labeled by an azido-sugar, a mass shift assay using a 5-kilodalton PEG mass tag carrying an alkynyl functional group may be performed to determine the stoichiometry of the O-GlcNAc modification on individual proteins. 76 The intensity of the shifted bands relative to the unshifted band allows for determination of O-GlcNAc stoichiometry. To characterize the protein glycosite(s), mass spectrometry-based proteomics has emerged as the primary mechanism for site-specific mapping of the O-GlcNAc modification site on individual proteins to the complex proteome.…”