Glycosylation is one of the most common protein modifications and is essential for cells. This modification is exceptionally complex because glycans are highly diverse and can be covalently bound to several amino acid residues in proteins through various configurations. There are two major types of protein glycosylation, i.e., N-linked glycosylation in which glycans are attached to the side chain of asparagine and O-linked glycosylation referring to glycans being bound to the side chains of serine and threonine. 1,2 Glycosylation plays vital roles in cells, including determination of protein folding, trafficking and stability, and regulation of nearly every extracellular activity such as cell-cell communication and cellmatrix interactions. 3,4 Aberrant protein glycosylation is directly related to multiple diseases, including cancer, neurodegenerative disorders, pulmonary diseases, blood disorders, and genetic diseases. 5,6 Due to the importance and complexity of protein glycosylation in biological systems, there is a longstanding interest to develop innovative methods to study glycoproteins and apply them for biomedical research. Investigation of protein glycosylation has become more popular with the development of modern instrumentation and computational methods. According to a PubMed search using the keyword "glycosylation", 16 publications were listed during 1960-1970 while over 20 000 studies were reported in the past 10 years. With the growing interests in protein glycosylation, this trend is expected to continue in the next decades.Mass spectrometry (MS)-based proteomics provides an excellent opportunity to globally analyze proteins and their modifications. [7][8][9][10][11][12][13][14][15][16][17][18][19] Nonetheless, it is still extremely challenging to comprehensively analyze protein glycosylation. 20 Unlike many other modifications with a fixed structure for the modified group, such as phosphorylation, the diversity of glycans makes it more challenging to employ the commonly used database searching methods such as SEQUEST and Mascot to identify glycopeptides in bottom-up proteomics. Lowabundance glycoproteins in complex biological samples are also hindered for detection by many high-abundance nonglycoproteins. Furthermore, glycans can interfere with the fragmentation of the peptide backbone. 20,21 Innovative and effective methods are critical to overcome these hurdles and to allow for comprehensive analysis of glycoproteins using MS.

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Protein glycosylation is ubiquitous in biological systems and plays essential roles in many cellular events. Global and site‐specific analysis of glycoproteins in complex biological samples can advance our understanding of glycoprotein functions and cellular activities. However, it is extraordinarily challenging because of the low abundance of many glycoproteins and the heterogeneity of glycan structures. The emergence of mass spectrometry (MS)‐based proteomics has provided us an excellent opportunity to comprehensively study proteins and their modifications, including glycosylation. In this review, we first summarize major methods for glycopeptide/glycoprotein enrichment, followed by the chemical and enzymatic methods to generate a mass tag for glycosylation site identification. We next discuss the systematic and quantitative analysis of glycoprotein dynamics. Reversible protein glycosylation is dynamic, and systematic study of glycoprotein dynamics helps us gain insight into glycoprotein functions. The last part of this review focuses on the applications of MS‐based proteomics to study glycoproteins in different biological systems, including yeasts, plants, mice, human cells, and clinical samples. Intact glycopeptide analysis is also included in this section. Because of the importance of glycoproteins in complex biological systems, the field of glycoproteomics will continue to grow in the next decade. Innovative and effective MS‐based methods will exponentially advance glycoscience, and enable us to identify glycoproteins as effective biomarkers for disease detection and drug targets for disease treatment. © 2019 Wiley Periodicals, Inc. Mass Spec Rev 9999: XX–XX, 2019.

Mass spectrometry (MS) has become an increasingly important technique to analyze proteins. In popular bottom-up MS-based proteomics, reduction and alkylation are routine steps to facilitate peptide identification. However, the reaction incompletion and side reactions may occur, which compromise the experimental results. In this work, we systematically evaluated the reduction step with the commonly used reagents, i.e., dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, or tris(3-hydroxypropyl)phosphine, and alkylation with iodoacetamide, acrylamide, N-ethylmaleimide, or 4-vinylpyridine. By using digested peptides from a yeast whole-cell lysate, the number of proteins and peptides identified were very similar using four different reducing reagents. The results from four alkylating reagents, however, were dramatically different with iodoacetamide giving the highest number of peptides with alkylated cysteine and the lowest number of peptides with incomplete cysteine alkylation and side reactions. Alkylation conditions with iodoacetamide were further optimized. To identify more peptides with cysteine, Thiopropyl-Sepharose 6B resins were used to enrich them, and the optimal conditions were employed for the reduction and alkylation. The enrichment resulted in over three times more cysteine-containing peptides than without enrichment. Systematic evaluation of the reduction and alkylation with different reagents can aid in a better design of bottom-up proteomic experiments.