Mass Spectrometric Determination of the Sites of O-Glycan Attachment with Low Picomolar Sensitivity
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文摘
A sensitive protocol for unambiguously and positively identifying O-glycosylation sites in glycopeptides is described, based on β-elimination of the glycan chain(s) using NH4OH. On glycan elimination, NH3is incorporated into the amino acid residue(s) to which the glycan(s) had been attached, to yield a modified amino acid residue having a distinct mass. Electrospray ionization collision-induced dissociation tandemmass spectrometry allows the released, modified peptide to be sequenced and the site(s) of the modified amino acid residue(s) to be identified. The protocol has been optimized using a series of structurally related O-glycopeptides, and standard conditions are recommended for handling unknowns. We demonstrate that site determination can be achieved using as little as 1 pmol of starting material.