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Altered protein glycosylation is typically associated with cognitive defects and other phenotypes, but there is a lack of knowledge about the brain glycoproteome. Here, we used the newly available O-glycoprotease IMPa from Pseudomonas aeruginosa for comprehensive O-glycoproteomic analyses of the mouse brain. In this approach, total tryptic glycopeptides were prepared, extracted, purified, and conjugated to a solid support before an enzymatic cleavage by IMPa. O-glycopeptides were analyzed by electron-transfer/higher-energy collision dissociation (EThcD), which permits site-specific and global analysis of all types of O-glycans. We developed two complementary approaches for the analysis of the total O-glycoproteome using HEK293 cells and derivatives. The results demonstrated that IMPa and EThcD facilitate the confident localization of O-glycans on glycopeptides. We then applied these approaches to characterize the O-glycoproteome of the mouse brain, which revealed the high frequency of various sialylated O-glycans along with the unusual presence of the Tn antigen. Unexpectedly, the results demonstrated that glycoproteins in the brain O-glycoproteome only partly overlap with those reported for the brain N-glycoproteome. These approaches will aid in identifying the novel O-glycoproteomes of different cells and tissues and foster clinical and translational insights into the functions of protein O-glycosylation in the brain and other organs.
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Encéfalo , Glicopeptídeos , Humanos , Animais , Camundongos , Células HEK293 , Transporte de Elétrons , ProteomaRESUMO
The subcellular localization of proteins is critical for their functions in eukaryotic cells and is tightly correlated with protein modifications. Here, we comprehensively investigate the nuclear-cytoplasmic distributions of the phosphorylated, O-GlcNAcylated, and non-modified forms of proteins to dissect the correlation between protein distribution and modifications. Phosphorylated and O-GlcNAcylated proteins have overall higher nuclear distributions than non-modified ones. Different distributions among the phosphorylated, O-GlcNAcylated, and non-modified forms of proteins are associated with protein size, structure, and function, as well as local environment and adjacent residues around modification sites. Moreover, we perform site-mutagenesis experiments using phosphomimetic and phospho-null mutants of two proteins to validate the proteomic results. Additionally, the effects of the OGT/OGA inhibition on glycoprotein distribution are systematically investigated, and the distribution changes of glycoproteins are related to their abundance changes under the inhibitions. Systematic investigation of the relationship between protein modification and localization advances our understanding of protein functions.
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Processamento de Proteína Pós-Traducional , Proteômica , Fosforilação , Proteômica/métodos , Glicoproteínas/metabolismo , Núcleo Celular/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/metabolismoRESUMO
GD2-targeting immunotherapies have improved survival in children with neuroblastoma, yet on-target, off-tumor toxicities can occur and a subset of patients cease to respond. The majority of neuroblastoma patients who receive immunotherapy have been previously treated with cytotoxic chemotherapy, making it paramount to identify neuroblastoma-specific antigens that remain stable throughout standard treatment. Cell surface glycoproteomics performed on human-derived neuroblastoma tumors in mice following chemotherapy treatment identified protein tyrosine kinase 7 (PTK7) to be abundantly expressed. Furthermore, PTK7 shows minimal expression on pediatric-specific normal tissues. We developed an anti-PTK7 chimeric antigen receptor (CAR) and find PTK7 CAR T cells specifically target and kill PTK7-expressing neuroblastoma in vitro. In vivo, human/murine binding PTK7 CAR T cells regress aggressive neuroblastoma metastatic mouse models and prolong survival with no toxicity. Together, these data demonstrate preclinical efficacy and tolerability for targeting PTK7 and support ongoing investigations to optimize PTK7-targeting CAR T cells for neuroblastoma.
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Neuroblastoma , Receptores de Antígenos Quiméricos , Humanos , Criança , Animais , Camundongos , Neuroblastoma/terapia , Neuroblastoma/patologia , Imunoterapia , Receptores de Antígenos Quiméricos/genética , Proteínas Tirosina QuinasesRESUMO
Manipulation of protein synthesis is commonly applied to uncover protein functions and cellular activities. Multiple inhibitors with distinct mechanisms have been widely investigated and employed in bio-related research, but it is extraordinarily challenging to measure and evaluate the synthesis inhibition efficiencies of individual proteins by different inhibitors at the proteome level. Newly synthesized proteins are the immediate and direct products of protein synthesis, and thus their comprehensive quantification provides a unique opportunity to study protein inhibition. Here, we systematically investigate protein inhibition and evaluate different popular inhibitors, i.e. cycloheximide, puromycin, and anisomycin, through global quantification of newly synthesized proteins in several types of human cells (A549, MCF-7, Jurkat, and THP-1 cells). The inhibition efficiencies of protein synthesis are comprehensively measured by integrating azidohomoalanine-based protein labeling, selective enrichment, a boosting approach, and multiplexed proteomics. The same inhibitor results in dramatic variation of the synthesis inhibition efficiencies for different proteins in the same cells, and each inhibitor exhibits unique preferences. Besides cell type- and inhibitor-specific effects, some universal rules are unraveled. For instance, nucleolar and ribosomal proteins have relatively higher inhibition efficiencies in every type of cells treated with each inhibitor. Moreover, proteins intrinsically resistant or sensitive to the inhibition are identified and found to have distinct functions. Systematic investigation of protein synthesis inhibition in several types of human cells by different inhibitors provides valuable information about the inhibition of protein synthesis, advancing our understanding of inhibiting protein synthesis.
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Surface and secreted glycoproteins are essential to cells and regulate many extracellular events. Because of the diversity of glycans, the low abundance of many glycoproteins, and the complexity of biological samples, a system-wide investigation of extracellular glycoproteins is a daunting task. With the development of modern mass spectrometry (MS)-based proteomics, comprehensive analysis of different protein modifications including glycosylation has advanced dramatically. This review focuses on the investigation of extracellular glycoproteins using MS-based proteomics. We first discuss the methods for selectively enriching surface glycoproteins and investigating protein interactions on the cell surface, followed by the application of MS-based proteomics for surface glycoprotein dynamics analysis and biomarker discovery. We then summarize the methods to comprehensively study secreted glycoproteins by integrating various enrichment approaches with MS-based proteomics and their applications for global analysis of secreted glycoproteins in different biological samples. Collectively, MS significantly expands our knowledge of extracellular glycoproteins and enables us to identify extracellular glycoproteins as potential biomarkers for disease detection and drug targets for disease treatment.
Assuntos
Glicoproteínas , Processamento de Proteína Pós-Traducional , Glicoproteínas/química , Glicosilação , Espectrometria de Massas/métodos , Proteômica/métodosRESUMO
Molecular changes in the brain of individuals afflicted with Alzheimer's disease (AD) are an intense area of study. Little is known about the role of protein abundance and posttranslational modifications in AD progression and treatment, in particular large-scale intact N-linked glycoproteomics analysis. To elucidate the N-glycoproteome landscape, we developed an approach based on multi-lectin affinity enrichment, hydrophilic interaction chromatography, and LC-MS-based glycoproteomics. We analyzed brain tissue from 10 persons with no cognitive impairment or AD, 10 with asymptomatic AD, and 10 with symptomatic AD, detecting over 300 glycoproteins and 1900 glycoforms across the samples. The majority of glycoproteins have N-glycans that are high-mannosidic or complex chains that are fucosylated and bisected. The Man5 N-glycan was found to occur most frequently at >20% of the total glycoforms. Unlike the glycoproteomes of other tissues, sialylation is a minor feature of the brain N-glycoproteome, occurring at <9% among the glycoforms. We observed AD-associated differences in the number of antennae, frequency of fucosylation, bisection, and other monosaccharides at individual glycosylation sites among samples from our three groups. Further analysis revealed glycosylation differences in subcellular compartments across disease stage, including glycoproteins in the lysosome frequently modified with paucimannosidic glycans. These results illustrate the N-glycoproteomics landscape across the spectrum of AD clinical and pathologic severity and will facilitate a deeper understanding of progression and treatment development.
Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Encéfalo/metabolismo , Proteoma/metabolismo , Polissacarídeos/metabolismoRESUMO
Protein O-GlcNAcylation plays critical roles in many cellular events, and its dysregulation is related to multiple diseases. Integrating bioorthogonal chemistry and multiplexed proteomics, we systematically and site specifically study the distributions and dynamics of protein O-GlcNAcylation in the nucleus and the cytoplasm of human cells. The results demonstrate that O-GlcNAcylated proteins with different functions have distinct distribution patterns. The distributions vary site specifically, indicating that different glycoforms of the same protein may have different distributions. Moreover, we comprehensively analyze the dynamics of O-GlcNAcylated and non-modified proteins in these two compartments, respectively, and the half-lives of glycoproteins in different compartments are markedly different, with the median half-life in the cytoplasm being much longer. In addition, glycoproteins in the nucleus are more dramatically stabilized than those in the cytoplasm under the O-GlcNAcase inhibition. The comprehensive spatial and temporal analyses of protein O-GlcNAcylation provide valuable information and advance our understanding of this important modification.
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Acetilglucosamina , Glicoproteínas , Proteômica , Acetilglucosamina/metabolismo , Citoplasma/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodosRESUMO
Glycoproteins on the surface of immune cells play extremely important roles in response to pathogens. Yet, a systematic and time-resolved investigation of surface glycoproteins during the immune response remains to be explored. Integrating selective enrichment of surface glycoproteins with multiplexed proteomics, we globally and site-specifically quantified the dynamics of surface glycoproteins on THP-1 monocytes and macrophages in response to bacterial infection and during the monocyte-to-macrophage differentiation. The time-resolved analysis reveals transient changes and differential remodeling of surface glycoproteins on both cell types, and potential upstream regulators and downstream effects of the regulated glycoproteins. Besides, we identified novel surface glycoproteins participating in the immune response such as APMAP, and site-specific changes of glycoproteins. This study provides unprecedented information to deepen our understanding of glycoproteins and cellular activities.
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The tyrosine residue of proteins participates in a wide range of activities including enzymatic catalysis, protein-protein interaction, and protein-ligand binding. However, the functional annotation of the tyrosine residues on a large scale is still very challenging. Here, we report a novel method integrating azo coupling, bioorthogonal chemistry, and multiplexed proteomics to globally investigate the tyrosine reactivity in the human proteome. Based on the azo-coupling reaction between aryl diazonium salt and the tyrosine residue, two different probes were evaluated, and the probe with the best performance was employed to further study the tyrosine residues in the human proteome. Then, tagged tyrosine-containing peptides were selectively enriched using bioorthogonal chemistry, and after the cleavage, a small tag on the peptides perfectly fits for site-specific analysis by MS. Coupling with multiplexed proteomics, we quantified over 5000 tyrosine sites in MCF7 cells, and these quantified sites displayed a wide range of reactivity. The tyrosine residues with high reactivity were found on functionally and structurally diverse proteins, including those with the catalytic activity and binding property. This method can be extensively applied to advance our understanding of protein functions and facilitate the development of covalent drugs to regulate protein activity.
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Proteoma , Tirosina , Humanos , Ligantes , Ligação Proteica , ProteômicaRESUMO
The cell plasma membrane provides a highly interactive platform for the information transfer between the inside and outside of cells. The surface glycoprotein interaction network is extremely important in many extracellular events, and aberrant protein interactions are closely correlated with various diseases including cancer. Comprehensive analysis of cell surface protein interactions will deepen our understanding of the collaborations among surface proteins to regulate cellular activity. In this work, we developed a method integrating chemical crosslinking, an enzymatic reaction, and MS-based proteomics to systematically characterize proteins interacting with surface glycoproteins, and then constructed the surfaceome interaction network. Glycans covalently bound to proteins were employed as "baits", and proteins that interact with surface glycoproteins were connected using chemical crosslinking. Glycans on surface glycoproteins were oxidized with galactose oxidase (GAO) and sequentially surface glycoproteins together with their interactors ("prey") were enriched through hydrazide chemistry. In combination with quantitative proteomics, over 300 proteins interacting with surface glycoproteins were identified. Many important domains related to extracellular events were found on these proteins. Based on the protein-protein interaction database, we constructed the interaction network among the identified proteins, in which the hub proteins play more important roles in the interactome. Through analysis of crosslinked peptides, specific interactors were identified for glycoproteins on the cell surface. The newly developed method can be extensively applied to study glycoprotein interactions on the cell surface, including the dynamics of the surfaceome interactions in cells with external stimuli.
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Glycoproteins on the surface of immune cells play extremely important roles in response to pathogens. Yet, a systematic and time-resolved investigation of surface glycoproteins during the immune response remains to be explored. Integrating selective enrichment of surface glycoproteins with multiplexed proteomics, we globally and site-specifically quantified the dynamics of surface glycoproteins on THP-1 monocytes and macrophages in response to bacterial infection and during the monocyte-to-macrophage differentiation. The time-resolved analysis reveals transient changes and differential remodeling of surface glycoproteins on both cell types, and potential upstream regulators and downstream effects of the regulated glycoproteins. Besides, we identified novel surface glycoproteins participating in the immune response such as APMAP, and site-specific changes of glycoproteins. This study provides unprecedented information to deepen our understanding of glycoproteins and cellular activities.
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Infecções Bacterianas/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/imunologia , Monócitos/imunologia , Humanos , Glicoproteínas de Membrana/químicaRESUMO
Glycoproteins secreted by cells play essential roles in the regulation of extracellular activities. Secreted glycoproteins are often reflective of cellular status, and thus glycoproteins from easily accessible bodily fluids can serve as excellent biomarkers for disease detection. Cultured cells have been extensively employed as models in the research fields of biology and biomedicine, and global analysis of glycoproteins secreted from these cells provides insights into cellular activities and glycoprotein functions. However, comprehensive identification and quantification of secreted glycoproteins is a daunting task because of their low abundances compared with the high-abundance serum proteins required for cell growth and proliferation. Several studies employed serum-free media to analyze secreted proteins, but it has been shown that serum starvation, even for a short period of time, can alter protein secretion. To overcome these issues, we developed a method to globally characterize secreted glycoproteins and their N-glycosylation sites from cultured cells by combining selective enrichment of secreted glycoproteins with a boosting approach. The results demonstrated the importance of the boosting sample selection and the boosting-to-sample ratio for improving the coverage of secreted glycoproteins. The method was applied to globally quantify secreted glycoproteins from THP-1 monocytes and macrophages in response to lipopolysaccharides (LPS) and from Hep G2 cells treated with TGF-ß without serum starvation. We found differentially secreted glycoproteins in these model systems that showed the cellular response to the immune activation or the epithelial-to-mesenchymal transition. Benefiting from the selective enrichment and the signal enhancement of low-abundance secreted glycoproteins, this method can be extensively applied to study secreted glycoproteins without serum starvation, which will provide a better understanding of protein secretion and cellular activity.
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Glicoproteínas/química , Técnicas de Cultura de Células , Química Click , Glicoproteínas/metabolismo , Células Hep G2 , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Peptídeos/química , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg2+ as a primary mediator of ribosomal structure and function. Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosome's early evolution in a high Fe2+ environment, and the continued use of Fe2+ by obligate anaerobes inhabiting high Fe2+ niches. Here, we show that (i) Fe2+ cleaves RNA by in-line cleavage, a non-oxidative mechanism that has not previously been shown experimentally for this metal, (ii) the first-order in-line rate constant with respect to divalent cations is >200 times greater with Fe2+ than with Mg2+, (iii) functional ribosomes are associated with Fe2+ after purification from cells grown under low O2 and high Fe2+ and (iv) a small fraction of Fe2+ that is associated with the ribosome is not exchangeable with surrounding divalent cations, presumably because those ions are tightly coordinated by rRNA and deeply buried in the ribosome. In total, these results expand the ancient role of iron in biochemistry and highlight a possible new mechanism of iron toxicity.
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Cátions Bivalentes/metabolismo , Ferro/metabolismo , Clivagem do RNA/genética , Ribossomos/genética , Sítios de Ligação , Cátions Bivalentes/química , Ferro/química , Magnésio/química , Magnésio/metabolismo , Metais/química , Metais/metabolismo , Oxirredução/efeitos dos fármacos , Ribossomos/químicaRESUMO
Protein synthesis is quickly and tightly regulated in cells to adapt to the ever-changing extracellular and intracellular environment. Accurate quantitation of rapid protein synthesis changes can provide insights into protein functions and cellular activities, but it is very challenging to achieve because of the lack of effective analysis methods. Here, we developed an effective mass spectrometry-based method named quantitative O-propargyl-puromycin tagging (QOT) by integrating O-propargyl-puromycin (OPP) labeling, bioorthogonal chemistry, and multiplexed proteomics for global and quantitative analysis of rapid protein synthesis. The current method enables us to accurately quantitate rapid changes of newly synthesized proteins because, unlike amino acids and their analogs, OPP can be utilized by the ribosome immediately without being activated and conjugated to tRNA, and thus cell starvation or pretreatment is not required. This method was applied to quantitate rapid changes of protein synthesis in THP-1 macrophages treated with lipopolysaccharide (LPS). For 15-min labeling, >3000 proteins were quantitated, and the synthesis of 238 proteins was significantly altered, including transcription factors and cytokines. The results demonstrated that protein synthesis was modulated to facilitate protein secretion in macrophages in response to LPS. Considering the importance of protein synthesis, this method can be extensively applied to investigate rapid changes of protein synthesis in the biological and biomedical research fields.
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Proteínas/análise , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas/síntese química , Proteômica , Puromicina/análogos & derivados , Puromicina/química , Células THP-1Assuntos
Glicopeptídeos/análise , Glicoproteínas/análise , Proteômica , Animais , Humanos , Espectrometria de MassasRESUMO
Glycation as a type of non-enzymatic protein modification is related to aging and chronic diseases, especially diabetes. Global analysis of protein glycation will aid in a better understanding of its formation mechanism and biological significance. In this work, we comprehensively investigated protein glycation in human cells (HEK293T, Jurkat, and MCF7 cells). The current results indicated that this non-enzymatic modification was not random, and protein at the extracellular regions and the nucleus were more frequently glycated. Systematic and site-specific analysis of glycated proteins allowed us to study the effect of the primary sequences and secondary structures of proteins on glycation. Furthermore, nearly every enzyme in the glycolytic pathway was found to be glycated and a possible mechanism was proposed. Many glycation sites were also previously reported as acetylation and ubiquitination sites, which strongly suggested that this non-enzymatic modification may disturb protein degradation and gene expression. The current results will facilitate further studies of protein glycation in biomedical and clinical research.
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Espectrometria de Massas/métodos , Proteínas/química , Acetilação , Sequência de Aminoácidos , Expressão Gênica , Glicólise , Glicosilação , Células HEK293 , Humanos , Células Jurkat , Células MCF-7 , Modelos Moleculares , Proteínas/genética , Proteólise , Proteômica/métodos , UbiquitinaçãoRESUMO
Proteins on the cell surface are frequently glycosylated, and they are essential for cells. Surface glycoproteins regulate nearly every extracellular event, but compared with global analysis of proteins, comprehensive and site-specific analysis of surface glycoproteins is much more challenging and dramatically understudied. Here, combining metabolic labeling, click-chemistry and enzymatic reactions, and mass spectrometry-based proteomics, we globally characterized surface glycoproteins from eight popular types of human cells. This integrative and effective method allowed for the identification of 2172 N-glycosylation sites and 1047 surface glycoproteins. The distribution and occurrence of N-glycosylation sites were systematically investigated, and protein secondary structures were found to have a dramatic influence on glycosylation sites. As expected, most sites are located on disordered regions. For the sites with the motif N-!P-C, about one-third of them are located on helix structures, while those with the motif N-!P-S/T prefer strand structures. There is almost no correlation between the number of glycosylation sites and protein length, but the number of sites corresponds well with the frequencies of the motif. Quantification results reveal that besides cell-specific glycoproteins, the uniqueness of each cell type further arises from differential expression of surface glycoproteins. The current research indicates that multiple surface glycoproteins including their abundances need to be considered for cell classification rather than a single cluster of differentiation (CD) protein normally used in conventional methods. These results provide valuable information to the glycoscience and biomedical communities and aid in the discovery of surface glycoproteins as disease biomarkers and drug targets.
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Glicoproteínas de Membrana/análise , Proteômica/métodos , Motivos de Aminoácidos , Antígenos CD/análise , Biotina/química , Linhagem Celular Tumoral , Separação Celular/métodos , Química Click , Glicosilação , Humanos , Espectrometria de Massas , Glicoproteínas de Membrana/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Estrutura Secundária de ProteínaRESUMO
rRNA is the single most abundant polymer in most cells. Mammalian rRNAs are nearly twice as large as those of prokaryotes. Differences in rRNA size are due to expansion segments, which contain extended tentacles in metazoans. Here we show that the terminus of an rRNA tentacle of Homo sapiens contains 10 tandem G-tracts that form highly stable G-quadruplexes in vitro. We characterized rRNA of the H. sapiens large ribosomal subunit by computation, circular dichroism, UV melting, fluorescent probes, nuclease accessibility, electrophoretic mobility shifts, and blotting. We investigated Expansion Segment 7 (ES7), oligomers derived from ES7, intact 28S rRNA, 80S ribosomes, and polysomes. We used mass spectrometry to identify proteins that bind to rRNA G-quadruplexes in cell lysates. These proteins include helicases (DDX3, CNBP, DDX21, DDX17) and heterogeneous nuclear ribonucleoproteins. Finally, by multiple sequence alignments, we observe that G-quadruplex-forming sequences are a general feature of LSU rRNA of Chordata but not, as far as we can tell, of other species. Chordata ribosomes present polymorphic tentacles with the potential to switch between inter- and intramolecular G-quadruplexes. To our knowledge, G-quadruplexes have not been reported previously in ribosomes.
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Quadruplex G , RNA Ribossômico/química , Animais , Sequência de Bases , Dicroísmo Circular , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Conformação de Ácido Nucleico , Subunidades Ribossômicas Maiores/química , Alinhamento de SequênciaRESUMO
The cell surface is normally covered with sugars that are bound to lipids or proteins. Surface glycoproteins play critically important roles in many cellular events, including cell-cell communications, cell-matrix interactions, and response to environmental cues. Aberrant protein glycosylation on the cell surface is often a hallmark of human diseases such as cancer and infectious diseases. Global analysis of surface glycoproteins will result in a better understanding of glycoprotein functions and the molecular mechanisms of diseases and the discovery of surface glycoproteins as biomarkers and drug targets. Here, an enzyme is exploited to tag surface glycoproteins, generating a chemical handle for their selective enrichment prior to mass spectrometric (MS) analysis. The enzymatic reaction is very efficient, and the reaction conditions are mild, which are well-suited for surface glycoprotein tagging. For biologically triplicate experiments, on average 953 N-glycosylation sites on 393 surface glycoproteins per experiment were identified in MCF7 cells. Integrating chemical and enzymatic reactions with MS-based proteomics, the current method is highly effective to globally and site-specifically analyze glycoproteins only located on the cell surface. Considering the importance of surface glycoproteins, this method is expected to have extensive applications to advance glycoscience.
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Membrana Celular/metabolismo , Galactose Oxidase/metabolismo , Marcação por Isótopo/métodos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Glicosilação , Humanos , Células MCF-7RESUMO
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.