Abstract
Despite increasing importance of protein glycosylation, most of the large-scale glycoproteomics have been limited to profiling the sites of N-glycosylation. However, in-depth knowledge of protein glycosylation to uncover functions and their clinical applications requires quantitative glycoproteomics eliciting both peptide and glycan sequences concurrently. Here we describe a novel strategy for the multiplexed quantitative mouse serum glycoproteomics based on a specific chemical ligation, namely, reverse glycoblotting technique, focusing sialic acids and multiple reaction monitoring (MRM). LC-MS/MS analysis of de-glycosylated peptides identified 270 mouse serum peptides (95 glycoproteins) as sialylated glycopeptides, of which 67 glycopeptides were fully characterized by MS/MS analyses in a straightforward manner. We revealed the importance of a fragment ion containing innermost N-acetylglucosamine (GlcNAc) residue as MRM transitions regardless the sequence of the peptides. Versatility of the reverse glycoblotting-assisted MRM assays was demonstrated by quantitative comparison of 25 targeted glycopeptides from 16 proteins between mice with homo and hetero types of diabetes disease model.
Highlights
Despite increasing importance of protein glycosylation, most of the large-scale glycoproteomics have been limited to profiling the sites of N-glycosylation
Selective, and quantitative oxidation of the tryptic digests of whole glycoproteins derived from 50 l of mouse serum by treating with 30 mM NaIO4 at 4 °C for 60 min, aldehydes generated at terminal sialic acid residues were selectively captured at 37 °C for 2 h by chemical ligation with commercially available hydrazide modified polymer (Affi-Gel Hz)
It is expected that enhanced ionization potency by pyridyl aminated (PA)-derivatization should greatly facilitate the structural characterization of sialyl glycopeptides through ESI-mass spectrometry (MS) and MS/MS analysis of ideal fragment ions
Summary
Despite increasing importance of protein glycosylation, most of the large-scale glycoproteomics have been limited to profiling the sites of N-glycosylation. We describe a novel strategy for the multiplexed quantitative mouse serum glycoproteomics based on a specific chemical ligation, namely, reverse glycoblotting technique, focusing sialic acids and multiple reaction monitoring (MRM). Targeted proteomics using multiple reaction monitoring (MRM) is emerging as a technology that complements the discovery capabilities of shotgun strategies as well as an alternative powerful novel MS-based approach to measure a series of candidate biomarkers [1,2,3,4,5,6,7]. Several such transitions (precursor/fragment ion pairs) are monitored over time, yielding a set of chromatographic traces with retention time and signal intensity for a specific transition as coordinates These measurements have been multiplexed to provide 30 or Reverse glycoblotting and MRM more specific assays in one run. Such methods are slowly gaining acceptance in the clinical laboratory for the routine measurement of endogenous metabolites [10] (e.g. in screening newborns for a panel of inborn errors of metabolism) some drugs [11] (e.g. immunosuppressants), and the component analysis of sugars [12]
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