AMPK activator-treated human cardiac spheres enhance maturation and enable pathological modeling.

BACKGROUND: Cardiac pathological outcome of metabolic remodeling is difficult to model using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) due to low metabolic maturation. METHODS: hiPSC-CM spheres were treated with AMP-activated protein kinase (AMPK) activators and examined for hiPSC-CM maturation features, molecular changes and the response to pathological stimuli. RESULTS: Treatment of hiPSC-CMs with AMPK activators increased ATP content, mitochondrial membrane potential and content, mitochondrial DNA, mitochondrial function and fatty acid uptake, indicating increased metabolic maturation. Conversely, the knockdown of AMPK inhibited mitochondrial maturation of hiPSC-CMs. In addition, AMPK activator-treated hiPSC-CMs had improved structural development and functional features-including enhanced Ca2+ transient kinetics and increased contraction. Transcriptomic, proteomic and metabolomic profiling identified differential levels of expression of genes, proteins and metabolites associated with a molecular signature of mature cardiomyocytes in AMPK activator-treated hiPSC-CMs. In response to pathological stimuli, AMPK activator-treated hiPSC-CMs had increased glycolysis, and other pathological outcomes compared to untreated cells. CONCLUSION: AMPK activator-treated cardiac spheres could serve as a valuable model to gain novel insights into cardiac diseases.

Systematic characterization of extracellular glycoproteins using mass spectrometry.

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.

Quantitative Structural Proteomics Unveils the Conformational Changes of Proteins under the Endoplasmic Reticulum Stress.

Protein structures are decisive for their activities and interactions with other molecules. Global analysis of protein structures and conformational changes cannot be achieved by commonly used abundance-based proteomics. Here, we integrated cysteine covalent labeling, selective enrichment, and quantitative proteomics to study protein structures and structural changes on a large scale. This method was applied to globally investigate protein structures in HEK293T cells and protein structural changes in the cells with the tunicamycin (Tm)-induced endoplasmic reticulum (ER) stress. We quantified several thousand cysteine residues, which contain unprecedented and valuable information of protein structures. Combining this method with pulsed stable isotope labeling by amino acids in cell culture, we further analyzed the folding state differences between pre-existing and newly synthesized proteins in cells under the Tm treatment. Besides newly synthesized proteins, unexpectedly, many pre-existing proteins were found to become unfolded upon ER stress, especially those related to gene transcription and protein translation. Furthermore, the current results reveal that N-glycosylation plays a more important role in the folding process of the tertiary and quaternary structures than the secondary structures for newly synthesized proteins. Considering the importance of cysteine in protein structures, this method can be extensively applied in the biological and biomedical research fields.

Carfilzomib Treatment Causes Molecular and Functional Alterations of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Background Anticancer therapies have significantly improved patient outcomes; however, cardiac side effects from cancer therapies remain a significant challenge. Cardiotoxicity following treatment with proteasome inhibitors such as carfilzomib is known in clinical settings, but the underlying mechanisms have not been fully elucidated. Methods and Results Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a cell model for drug-induced cytotoxicity in combination with traction force microscopy, functional assessments, high-throughput imaging, and comprehensive omic analyses, we examined the molecular mechanisms involved in structural and functional alterations induced by carfilzomib in hiPSC-CMs. Following the treatment of hiPSC-CMs with carfilzomib at 0.01 to 10 µmol/L, we observed a concentration-dependent increase in carfilzomib-induced toxicity and corresponding morphological, structural, and functional changes. Carfilzomib treatment reduced mitochondrial membrane potential, ATP production, and mitochondrial oxidative respiration and increased mitochondrial oxidative stress. In addition, carfilzomib treatment affected contractility of hiPSC-CMs in 3-dimensional microtissues. At a single cell level, carfilzomib treatment impaired Ca2+ transients and reduced integrin-mediated traction forces as detected by piconewton tension sensors. Transcriptomic and proteomic analyses revealed that carfilzomib treatment downregulated the expression of genes involved in extracellular matrices, integrin complex, and cardiac contraction, and upregulated stress responsive proteins including heat shock proteins. Conclusions Carfilzomib treatment causes deleterious changes in cellular and functional characteristics of hiPSC-CMs. Insights into these changes could be gained from the changes in the expression of genes and proteins identified from our omic analyses.

Chronic Ethanol Exposure Induces Deleterious Changes in Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.

Chronic alcohol consumption in adults can induce cardiomyopathy, arrhythmias, and heart failure. In newborns, prenatal alcohol exposure can increase the risk of congenital heart diseases. Understanding biological mechanisms involved in the long-term alcohol exposure-induced cardiotoxicity is pivotal to the discovery of therapeutic strategies. In this study, cardiomyocytes derived from human pluripotent stem cells (hiPSC-CMs) were treated with clinically relevant doses of ethanol for various durations up to 5 weeks. The treated cells were characterized for their cellular properties and functions, and global proteomic profiling was conducted to understand the molecular changes associated with long-term ethanol exposure. Increased cell death, oxidative stress, deranged Ca2+ handling, abnormal action potential, altered contractility, and suppressed structure development were observed in ethanol-treated cells. Many dysregulated proteins identified by global proteomic profiling were involved in apoptosis, heart contraction, and extracellular collagen matrix. In addition, several signaling pathways including the Wnt and TGFß signaling pathways were affected due to long-term ethanol treatment. Therefore, chronic ethanol treatment of hiPSC-CMs induces cardiotoxicity, impairs cardiac functions, and alters protein expression and signaling pathways. This study demonstrates the utility of hiPSC-CMs as a novel model for chronic alcohol exposure study and provides the molecular mechanisms associated with long-term alcohol exposure in human cardiomyocytes.

An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome.

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.

Unraveling the surface glycoprotein interaction network by integrating chemical crosslinking with MS-based proteomics.

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.

Transcriptional firing represses bactericidal activity in cystic fibrosis airway neutrophils.

Neutrophils are often considered terminally differentiated and poised for bacterial killing. In chronic diseases such as cystic fibrosis (CF), an unexplained paradox pits massive neutrophil presence against prolonged bacterial infections. Here, we show that neutrophils recruited to CF airways in vivo and in an in vitro transmigration model display rapid and broad transcriptional firing, leading to an upregulation of anabolic genes and a downregulation of antimicrobial genes. Newly transcribed RNAs are mirrored by the appearance of corresponding proteins, confirming active translation in these cells. Treatment by the RNA polymerase II and III inhibitor α-amanitin restores the expression of key antimicrobial genes and increases the bactericidal capacity of CF airway neutrophils in vitro and in short-term sputum cultures ex vivo. Broadly, our findings show that neutrophil plasticity is regulated at the site of inflammation via RNA and protein synthesis, leading to adaptations that affect their canonical functions (i.e., bacterial clearance).

MS-based proteomics for comprehensive investigation of protein O-GlcNAcylation.

Protein O-GlcNAcylation refers to the covalent binding of a single N-acetylglucosamine (GlcNAc) to the serine or threonine residue. This modification primarily occurs on proteins in the nucleus and the cytosol, and plays critical roles in many cellular events, including regulation of gene expression and signal transduction. Aberrant protein O-GlcNAcylation is directly related to human diseases such as cancers, diabetes and neurodegenerative diseases. In the past decades, considerable progress has been made for global and site-specific analysis of O-GlcNAcylation in complex biological samples using mass spectrometry (MS)-based proteomics. In this review, we summarized previous efforts on comprehensive investigation of protein O-GlcNAcylation by MS. Specifically, the review is focused on methods for enriching and site-specifically mapping O-GlcNAcylated peptides, and applications for quantifying protein O-GlcNAcylation in different biological systems. As O-GlcNAcylation is an important protein modification for cell survival, effective methods are essential for advancing our understanding of glycoprotein functions and cellular events.

Enhancing Comprehensive Analysis of Secreted Glycoproteins from Cultured Cells without Serum Starvation.

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.

Melphalan induces cardiotoxicity through oxidative stress in cardiomyocytes derived from human induced pluripotent stem cells.

BACKGROUND: Treatment-induced cardiotoxicity is a leading noncancer-related cause of acute and late onset morbidity and mortality in cancer patients on antineoplastic drugs such as melphalan-increasing clinical case reports have documented that it could induce cardiotoxicity including severe arrhythmias and heart failure. As the mechanism by which melphalan impairs cardiac cells remains poorly understood, here, we aimed to use cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) to investigate the cellular and molecular mechanisms of melphalan-induced cardiotoxicity. METHODS: hiPSC-CMs were generated and treated with clinically relevant doses of melphalan. To characterize melphalan-induced cardiotoxicity, cell viability and apoptosis were quantified at various treatment durations. Ca2+ transient and contractility analyses were used to examine the alterations of hiPSC-CM function. Proteomic analysis, reactive oxygen species detection, and RNA-Sequencing were conducted to investigate underlying mechanisms. RESULTS: Melphalan treatment of hiPSC-CMs induced oxidative stress, caused Ca2+ handling defects and dysfunctional contractility, altered global transcriptomic and proteomic profiles, and resulted in apoptosis and cell death. The antioxidant N-acetyl-L-cysteine attenuated these genomic, cellular, and functional alterations. In addition, several other signaling pathways including the p53 and transforming growth factor-ß signaling pathways were also implicated in melphalan-induced cardiotoxicity according to the proteomic and transcriptomic analyses. CONCLUSIONS: Melphalan induces cardiotoxicity through the oxidative stress pathway. This study provides a unique resource of the global transcriptomic and proteomic datasets for melphalan-induced cardiotoxicity and can potentially open up new clinical mechanism-based targets to prevent and treat melphalan-induced cardiotoxicity.

Proteomic Profiling Reveals Roles of Stress Response, Ca2+ Transient Dysregulation, and Novel Signaling Pathways in Alcohol-Induced Cardiotoxicity.

BACKGROUND: Alcohol use in pregnancy increases the risk of abnormal cardiac development, and excessive alcohol consumption in adults can induce cardiomyopathy, contractile dysfunction, and arrhythmias. Understanding molecular mechanisms underlying alcohol-induced cardiac toxicity could provide guidance in the development of therapeutic strategies. METHODS: We have performed proteomic and bioinformatic analysis to examine protein alterations globally and quantitatively in cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) treated with ethanol (EtOH). Proteins in both cell lysates and extracellular culture media were systematically quantitated. RESULTS: Treatment with EtOH caused severe detrimental effects on hiPSC-CMs as indicated by significant cell death and deranged Ca2+ handling. Treatment of hiPSC-CMs with EtOH significantly affected proteins responsible for stress response (e.g., GPX1 and HSPs), ion channel-related proteins (e.g. ATP1A2), myofibril structure proteins (e.g., MYL2/3), and those involved in focal adhesion and extracellular matrix (e.g., ILK and PXN). Proteins involved in the TNF receptor-associated factor 2 signaling (e.g., CPNE1 and TNIK) were also affected by EtOH treatment. CONCLUSIONS: The observed changes in protein expression highlight the involvement of oxidative stress and dysregulation of Ca2+ handling and contraction while also implicating potential novel targets in alcohol-induced cardiotoxicity. These findings facilitate further exploration of potential mechanisms, discovery of novel biomarkers, and development of targeted therapeutics against EtOH-induced cardiotoxicity.

A Chemoenzymatic Method Based on Easily Accessible Enzymes for Profiling Protein O-GlcNAcylation.

O-GlcNAcylation has gradually been recognized as a critically important protein post-translational modification in mammalian cells. Besides regulation of gene expression, its crosstalk with protein phosphorylation is vital for cell signaling. Despite its importance, comprehensive analysis of O-GlcNAcylation is extraordinarily challenging due to the low abundances of many O-GlcNAcylated proteins and the complexity of biological samples. Here, we developed a novel chemoenzymatic method based on a wild-type galactosyltransferase and uridine diphosphate galactose (UDP-Gal) for global and site-specific analysis of protein O-GlcNAcylation. This method integrates enzymatic reactions and hydrazide chemistry to enrich O-GlcNAcylated peptides. All reagents used are more easily accessible and cost-effective as compared to the engineered enzyme and click chemistry reagents. Biological triplicate experiments were performed to validate the effectiveness and the reproducibility of this method, and the results are comparable with the previous chemoenzymatic method using the engineered enzyme and click chemistry. Moreover, because of the promiscuity of the galactosyltransferase, 18 unique O-glucosylated peptides were identified on the EGF domain from nine proteins. Considering that effective and approachable methods are critical to advance glycoscience research, the current method without any sample restrictions can be widely applied for global analysis of protein O-GlcNAcylation in different samples.

Systematic and site-specific analysis of N-glycoproteins on the cell surface by integrating bioorthogonal chemistry and MS-based proteomics.

Glycoproteins on the cell surface are essential for various cellular activities including cell-cell communication and cell-matrix interaction. Alterations of glycosylation are correlated with many diseases such as cancer and infectious diseases. However, it is greatly challenging to systematically and site-specially analyze glycoproteins only located on cell surface because of the heterogeneity of glycans, the low abundance of many surface glycoproteins and the requirement of effective methods to separate surface glycoproteins. In this chapter, we briefly review existing mass spectrometry (MS)-based methods for global analysis of surface glycoproteins. Then we discuss an effective method integrating metabolic labeling, click and enzymatic reactions, and MS-based proteomics to comprehensively and site-specifically investigate cell surface N-glycoproteins. A detailed protocol for this method is also included. In combination with quantitative proteomics, we applied this method to quantify cell surface N-glycoproteins and study the relationship between cell invasiveness and N-sialoglycoproteins on the cell surface. Considering the importance of surface glycoproteins, this method can be extensively applied to advance glycoscience, which leads to a better understanding of the molecular mechanisms of human diseases, and the discovery of surface glycoproteins as biomarkers for disease detection.

Comprehensive Analysis of Protein Glycation Reveals Its Potential Impacts on Protein Degradation and Gene Expression in Human Cells.

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.

Surface Glycoproteomic Analysis Reveals That Both Unique and Differential Expression of Surface Glycoproteins Determine the Cell Type.

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.

Enzymatic Tagging of Glycoproteins on the Cell Surface for Their Global and Site-Specific Analysis with Mass Spectrometry.

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.

Global and site-specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry.

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-Based Chemical and Enzymatic Methods for Global Analysis of Protein Glycosylation.

Glycosylation is one of the most common protein modifications, and it is essential for mammalian cell survival. It often determines protein folding and trafficking, and regulates nearly every extracellular activity, including cell-cell communication and cell-matrix interactions. Aberrant protein glycosylation events are hallmarks of human diseases such as cancer and infectious diseases. Therefore, glycoproteins can serve as effective biomarkers for disease detection and targets for drug and vaccine development. Despite the importance of glycoproteins, global analysis of protein glycosylation (either glycoproteins or glycans) in complex biological samples has been a daunting task, and here we mainly focus on glycoprotein analysis using mass spectrometry (MS)-based bottom-up proteomics. Although the emergence of MS-based proteomics has provided a great opportunity to analyze glycoproteins globally, the low abundance of many glycoproteins and the heterogeneity of glycans dramatically increase the technical difficulties. In order to overcome these obstacles, considerable progress has been made in recent years, which has contributed to comprehensive analysis of glycoproteins. In our lab, we developed effective MS-based chemical and enzymatic methods to (1) globally analyze glycoproteins in complex biological samples, (2) target glycoproteins specifically on the surface of human cells, (3) systematically quantify glycoprotein and surface glycoprotein dynamics (the abundance changes of glycoproteins as a function of time), and (4) selectively characterize glycoproteins with a particular and important glycan. In this Account, we first briefly describe the glycopeptide/protein enrichment methods in the literature and then discuss the developments of boronic acid-based methods to enrich glycopeptides for large-scale analysis of protein glycosylation. Boronic acids can form reversible covalent interactions with sugars, but the low binding affinity of normal boronic acid-based methods prevents us from capturing glycoproteins with low abundance, which often contain more valuable information. We enhanced the boronic acid-glycan interactions by using a boronic acid derivative (benzoboroxole) and conjugating it onto a dendrimer to allow synergistic interactions between the boronic acid derivative and sugars. The new method is capable of globally analyzing protein glycosylation with site and glycan structure information, especially for those with low abundance. In the next part, we discuss the combination of metabolic labeling, click chemistry and enzymatic reactions, and MS-based proteomics as a very powerful approach for surface glycoproteome analysis in human cells. The methods enable us to specifically identify surface glycoproteins and to quantify their abundance changes and dynamics together with quantitative proteomics. The last section of this Account focuses on chemical and enzymatic methods to study glycoproteins containing a particular and important glycan (the Tn antigen, i.e., O-GalNAc). Although not comprehensive, this Account provides an overview of chemical and enzymatic methods to characterize protein glycosylation in combination with MS-based proteomics. These methods will have extensive applications in the fields of biology and biomedicine, which will lead to a better understanding of glycoprotein functions and the molecular mechanisms of diseases. Eventually, glycoproteins will be identified as effective biomarkers for disease detection and drug targets for disease treatment.