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.