Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry

Abstract

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.

Figure 1

(a) Structure of the Amadori compound (AC) resulting from reaction of primary amino groups with D-glucose; (b) structure of m-aminophenylboronic affinity gel, which is covalently bound to agarose as the solid matrix; and (c) the equilibria between boronic acids and cis-diol-containing compounds. Affinity attachment, elution and regeneration of boronic acid are shown.

Figure 2

(a) Chromatograms of different standard proteins passing through the Glycogel^™ II boronate affinity column. The traces are labeled with the respective sample name, and the separation conditions are shown in the experimental section. Peaks at approximately 2 min are non-glycated proteins, and glycated proteins elute near 15 min; (b) chromatogram showing the enrichment of glycated peptides from a tryptic digest of enriched glycated RNase. The glycated peptides elute slightly before 15 min, and the non-glycated peptides are washed out near 2 min.

Figure 3

(a) Chromatogram showing the enrichment of glycated proteins from human serum glycated in vitro; (b) chromatogram showing the enrichment of glycated peptides from tryptic digest of glycated proteins (dotted line) obtained in (a) and non-enriched glycated human serum (solid line). The separation conditions are shown in the experimental section. The chromatograms were normalized based on the most intense peak in each trace, i.e. the peak corresponding to the flow-through fraction.

Figure 4

MS/MS spectra obtained under CID (a) and ETD (b) fragmentation modes respectively of m/z 499.6, the [M + 3H]³⁺ of peptide LVDkFLEDVKK from α-1-antitrypsin precursor, in which “k” represents lysine modification with glucose. Inset in (a) is the zoom in view of the ions between m/z 445.5 and m/z 487.4; the identified c and z ions were labeled above and below the sequence in (b).