Advantages of pyrene derivatization to site-specific glycosylation analysis on MALDI mass spectrometry

Abstract

Glycoproteomics involving the analysis of glycopeptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a new and attractive technique. However, quantitative performance in MALDI-MS is hampered by its poor reproducibility among laser shots. 2,5-Dihydroxybenzoic acid (DHBA) is a useful matrix for glycopeptides but forms highly heterogeneous crystals. In this study, we have investigated the distribution of significant signals generated from a sample of glycopeptides on the target plate using a MALDI imaging technique. MALDI images of glycopeptides, which have different glycans on the same peptide, in the Lys-C digests of bovine ribonuclease B were identical. Thus, all glycoforms on a given peptide can be detected at the same laser irradiation spot simultaneously, which offers a significant advantage over other techniques. A similar result was observed with glycopeptides of human serum immunoglobulin G. Interestingly, distinct MALDI images were observed for glycopeptides having different amino acid sequences, despite having an identical glycan structure. The common peptides, which were glycosylated or non-glycosylated, or sialylated or desialylated gave similar MALDI images. Taken together, our results suggest that sweet spot localization of glycopeptides is dependent on the peptide moiety rather than the glycan structure. Furthermore, introduction of pyrene group to glycopeptides which have different peptides result in a uniform MALDI image. It suggested that pyrene derivatization in MALDI-MS facilitates straightforward analysis of a glycopeptide mixture because the same mass spectrum can be obtained at every sweet spot in addition to increase in signal intensity. Thus, this study validates the use of MALDI-MS for site-specific glycoprofiling at the glycopeptide level.