Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry - PubMed (original) (raw)

Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry

Zihao Wang et al. Mol Cell Proteomics. 2010 Jan.

Abstract

Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked beta-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.

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Figures

Fig. 1.

Fig. 1.

Enrichment of _O_-GlcNAc-modified peptides by a combination of enzymatic labeling with UDP-GalNAz and chemical derivatization with the photocleavable reagent PC-PEG-biotin-alkyne. a, flow chart showing the overall strategy. Inset, structure of the photocleavable PC-PEG-biotin-alkyne reagent. CIP, calf intestine phosphatase; PNGase F, peptide:_N_-Glycosidase F; TBTA, tris[(1-benzyl-1_H_-1,2,3-triazol-4-yl) methyl]amine. b, CAD mass spectrum of a tagged synthetic _O_-GlcNAc-modified peptide, YSPTgSPSK (gS is _O_-GlcNAcylated Ser), showing signature ions at m/z 300.2 and 503.1 that result from cleavage at the two sugar ketal linkages and that confirm the presence of the tagged _O_-GlcNAc moiety. c, ETD mass spectrum recorded on [M + 3H]3+ (m/z 457.2) ions from the same peptide, YSPTgSPSK. Predicted m/z values for ions of type c′ and z′· (monoisotopic and average masses for singly and doubly charged ions, respectively) are shown above and below the peptide sequence. Observed product ions are underlined and also labeled in the spectrum. Ions in the precursor isolation window are labeled with a triangle (▾). Brackets enclose ions that correspond to charge-reduced species and fragments derived from them by loss of small, neutral molecules. Product ions that result from loss of an aminomethyltriazole radical are labeled with a circle (○). M represents the tagged peptide.

Fig. 2.

Fig. 2.

Characterization of an _O_-GlcNAc-modified peptide from α-crystallin. a, CAD mass spectrum showing signature ions characteristic of the derivatized _O_-GlcNAc moiety. M represents the tagged peptide. b, ETD mass spectrum recorded on [M + 4H]4+ ions (m/z 537.10) from the same peptide, AIPVgSREEKPSSAPSS. Predicted m/z values for ions of type c′ and z′· (monoisotopic masses and average masses for singly and doubly charged ions, respectively) are shown above and below the peptide sequence. Observed product ions are underlined and also labeled in the spectrum. Ions in the precursor isolation window are labeled with a triangle (▾). Brackets enclose ions that correspond to charge-reduced species and fragments derived from them by loss of small, neutral molecules. Product ions that result from loss of an aminomethyltriazole radical are labeled with a circle (○). c, the tagging approach enables detection of _O_-GlcNAc-modified proteins by avidin-HRP blotting. This figure shows that tagged α-crystalline provides a strong signal after blotting with avidin-HRP blotting. Upon UV illumination, no signal is observed indicating photocleavage and loss of the biotin tag from α-crystallin.

Fig. 3.

Fig. 3.

Enriched and tagged _O_-GlcNAc-modified peptides from tau-containing protein fractions isolated from rat brain. a, ETD MS/MS spectrum recorded on [M + 3H]3+ ions (m/z 474.2) for the derivatized peptide VVgSDTSPR from the tau protein. b, ETD MS/MS spectrum recorded on [M + 3H]3+ ions (m/z 748.1) for the derivatized peptide VAAAAgTTTTTTTTTVAEK from MeCP2. Spectra are labeled as described in Figs. 1_c_ and 2_b_.

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References

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