Recent advances in glycopeptide enrichment and mass spectrometry data interpretation approaches for glycoproteomics analyses

Protein glycosylation is one of the most important post-translational modifications (PTMs). The glycosylation is crucial in a variety of physiological and pathological processes that include protein stability, intracellular and intercellular signal transduction, hormone activation or inactivation, and immune regulation. Protein glycosylation is generated by complex biosynthetic pathways comprising hundreds of glycosyltransferases, glycosidases, transcriptional factors, transporters, and protein backbones. Abnormal protein glycosylation is closely associated with the occurrence and development of diseases. Many disease biomarkers in clinical screening are glycoproteins (alfa fetoprotein for liver cancer, carbohydrate antigen 125 for ovarian cancer, carcinoembryonic antigen for colon cancer, prostate-specific antigen for prostate cancer, etc.), and glycan antigens (carbohydrate antigen 19-9 for gastrointestinal cancer and pancreatic cancer, etc.). Glycoproteomics research and technological developments are important to elucidate the mechanism of protein glycosylation in vivo. Mass spectrometry (MS)-based proteomics provides an excellent approach for the comprehensive analysis of proteins and their modifications. In bottom-up proteomics, glycoproteomic analysis is more difficult than other PTMs because intact glycopeptides have diverse peptide backbones and glycan chains, relatively low abundance and ionization efficiency, and pronounced heterogeneity. In recent years, glycoproteomic methodologies such as intact glycopeptide enrichment methods, MS fragmentation and acquisition approaches, MS data interpretation tools and software, and quantification strategies have been appreciably improved. These methodologies have driven in-depth glycoproteomics research. This review focuses on the recent advances in MS-based glycoproteomics. New enrichment methods and spectral interpretation approaches of intact N- and O-glycopeptides are discussed. Their applications in answering various questions in complex biological systems are also considered. The new enrichment methods for intact glycopeptides are mostly based on existing principles. Some properties of the materials, such as hydrophilicity and electrophilicity, have been optimized to improve the enrichment performance. For example, dual-functional Ti(IV)-IMAC materials have been used for the separation of glycopeptides and phosphopeptides. Considering the clinical applications, some glycoproteomics methods integrate enrichment processing into automated workflows to reduce errors caused by manual operations and to increase the experimental reproducibility and efficiency. For example, an automated glycopeptide enrichment method consisting of a liquid chromatograph equipped with a hydrophilic interaction chromatography column has been shown capable of highly reproducible analyses of site-specific glycopeptides in complex biological samples. These methods are more suitable for the discovery of newly glycosylation-related biomarkers as well as for the physiopathological studies of human diseases. With the optimization of glycopeptide enrichment methods and the innovation of MS technologies in the past decade, MS analysis of intact glycopeptides has begun to yield a wealth of glycopeptide fragment ions and plentiful high-quality MS data. This review introduces several effective fragmentation methods for intact glycopeptides. These include collision-induced dissociation, high-energy collision dissociation, electron capture dissociation, electron-transfer dissociation, and electron-transfer/higher-energy collision dissociation. Automated analysis of MS data of intact N- and O-glycopeptides requires interpretation approaches and corresponding software tools with high sensitivity and reliability. Finally, we highlight the utility of several spectral interpretation approaches and their corresponding popular search software, including ArMone, Byonic, GPQuest, pGlyco, O-search, MSFragger-Glyco, and O-Pair Search. In addition, MS data acquisition modes, such as data-dependent acquisition, data-independent acquisition, multiple reaction monitoring technology, and parallel reaction monitoring technology, have great application prospects in glycoproteomics research. With the improvements in enrichment methods, MS technologies, and spectral interpretation approaches for intact N- and O-glycopeptides, comprehensive and systematic glycoproteomics analysis has tremendously expanded the knowledge of protein glycosylation. These glycoproteomic technologies have a wide range of applications that include exploring the molecular mechanisms of protein glycosylation and discovering the new biomarkers of human diseases.