Synthesis of a Highly Azide-Reactive and Thermosensitive Biofunctional Reagent for Efficient Enrichment and Large-Scale Identification of O-GlcNAc Proteins by Mass Spectrometry

Abstract

O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification of proteins in eukaryotic cells. Despite their low abundance, O-GlcNAc-modified proteins play many important roles in regulating gene expression, signal transduction, and cell cycle. Aberrant O-GlcNAc proteins are correlated with many major human diseases, such as Alzheimer's disease, diabetes, and cancer. Because of the extremely low stoichiometry of O-GlcNAc proteins, enrichment is required before mass spectrometry analysis for large-scale identification and in-depth understanding of their cellular function. In this work, we designed and synthesized a novel thermosensitive immobilized triarylphosphine reagent as a convenient tool for efficient enrichment of azide-labeled O-GlcNAc proteins from complex biological samples. Immobilization of triarylphosphine on highly water-soluble thermosensitive polymer largely increases its solubility and reactivity in aqueous solution. As a result, facilitated coupling is achieved between triarylphosphine and azide-labeled O-GlcNAc proteins via Staudinger ligation, due to the increased triarylphosphine concentration, reduced interfacial mass transfer resistance, and steric hindrance in homogeneous reaction. Furthermore, solubility of the polymer from complete dissolution to full precipitation can be easily controlled by simply adjusting the environmental temperature. Therefore, facile sample recovery can be achieved by increasing the temperature to precipitate the polymer-O-GlcNAc protein conjugates from solution. This novel immobilized triarylphosphine reagent enables efficient enrichment and sensitive detection of more than 1700 potential O-GlcNAc proteins from HeLa cell using mass spectrometry, demonstrating its potential as a general strategy for low-abundance target enrichment.

O-GlcNAcylation Quantification of Certain Protein by the Proximity Ligation Assay and Clostridium perfringen OGAD298N(CpOGAD298N)

“…The top 20 significantly enriched functional groups are listed in Figure E. Go annotation analysis of the enriched proteins indicated that O-GlcNAc modified proteins are implicated in a large scale of cellular pathways and molecular functions, as reported in the previous proteomic analysis. − …”

Analytical and Biochemical Perspectives of Protein O-GlcNAcylation

“…Besides biotin–alkyne linkers, triarylphosphine derivatives, which readily react with azide-labeled molecules via Staudinger ligation, represent another alternative for O-GlcNAc enrichment. , Huo et al synthesized a triphenylphosphine-functionalized trimethylpiperidine (TFT) reagent consisting of three parts: a “click” group (toward azide-tagged peptides), a linker, and an affinity group mimicking TMT (which can reversibly bind to an anti-TMT antibody) (Figure C) . With this approach, the TFT-tagged O-GlcNAc peptides were enriched by using the anti-TMT antibody-conjugated agarose beads with a moderately high affinity ( K d = 86 pM) and then recovered by competitive elution under mild conditions.…”

The Staudinger Ligation

“…The entries are organized by number of carbon atoms of the phosphane (and then grouped by publication), and it will cover examples up to beginning of 2020 (for not or only partly covered examples, see refs − ).…”