Mass Spectrometry-based Strategies and Methods for N-linked Intact Glycopeptide Analysis

As one of the most common and important protein modifications, glycosylation has been one of the focuses of the proteomic researches. In the last decades, most of N-linked glycoproteomic studies focused mainly on the analysis of either released glycans or de-glycosylated peptides. While this strategy reduced the complexity of glycoprotein analysis, it lost glycosite-specific glycosylation information. Several strategies and methods for intact N-glycopeptide analysis have been established during the last few years. Generally, to achieve the identification and quantification of intact glycopeptides, the first step is to enrich glycopeptides from complex samples to. reduce the affects from non-glycosylated peptides, then the mass spectrometry parameter settings need to be adjusted to satisfy the fragment features of glycopeptides, importantly the related software also need to be developed for the precise identification of the peptide sequence and glycan structures or compositions of the intact glycopeptides. These three main aspects of the strategies for mass spectrometry-based intact glycopeptide analysis are discussed in this paper. Some further details, such as the recognition of intact glycopeptide spectra, precursor monoisotopic mass correction, database selection, as well as the false discovery rate (FDR) evaluation and control, are further discussed. Direct intact glycopeptide analyses, with the recovery of the glycosite-specific glycosylation information, will provides a powerful tool for biomarker discovery and the mechanism studies on various diseases.