Protein glycosylation is a highly heterogeneous post-translational modification that has been demonstrated to exhibit significant variations in various diseases. Due to the differential patterns observed in disease and healthy populations, the glycosylated proteins hold promise as early indicators for multiple diseases. With the continuous development of liquid chromatography-mass spectrometry (LC-MS) technology and spectrum analysis software, the sensitivity for the decipher of the tandem mass spectra of the glycopeptides carrying intact glycans, i.e., intact glycopeptides, enzymatic hydrolyzed from glycoproteins has been significantly improved. From quantified intact glycopeptides, the difference of protein glycosylation at multiple levels, e.g., glycoprotein, glycan, glycosite, and site-specific glycans, could be obtained for different samples. However, the manual analysis of the intact glycopeptide quantitative data at multiple levels is tedious and time consuming. In this study, we have developed a software tool named \"GP-Marker\" to facilitate large-scale data mining of spectra dataset of intact N-glycopeptide at multiple levels. This software provides a user-friendly and interactive interface, offering operational tools for machine learning to researchers without programming backgrounds. It includes a range of visualization plots displaying differential glycosylation and provides the ability to extract multi-level data analysis from intact glycopeptide data quantified by Glyco-Decipher.

Protein glycosylation is one of the most common and important post-translational modifications of proteins involved in regulating glycoprotein functions. The chemoenzymatic glycan labeling strategy allows rapid, efficient, and selective interrogation of glycoproteins. Glycoproteomics identifies protein glycosylation events at a large scale, providing information such as peptide sequences, glycan structures, and glycosylated sites. This review discusses the recent development of chemoenzymatic labeling strategies for glycoprotein analysis, mainly including glycoprotein and glycosite profiling. Furthermore, we highlight the chemoenzymatic enrichment approaches in mass spectrometry analysis for three classes of glycan modifications, including N-glycosylation, O-GlcNAcylation, and mucin-type O-glycosylation. Finally, we highlight the emerging trends in new tools and cutting-edge technologies available for glycoproteomic research.

Cell-surface proteins are extremely important for many cellular events, such as regulating cell-cell communication and cell-matrix interactions. Aberrant alterations in surface protein expression, modification (especially glycosylation), and interactions are directly related to human diseases. Systematic investigation of surface proteins advances our understanding of protein functions, cellular activities, and disease mechanisms, which will lead to identifying surface proteins as disease biomarkers and drug targets.
In this review, we summarize mass spectrometry (MS)-based proteomics methods for global analysis of cell-surface proteins. Then, investigations of the dynamics of surface proteins are discussed. Furthermore, we summarize the studies for the surfaceome interaction networks. Additionally, biological applications of MS-based surfaceome analysis are included, particularly highlighting the significance in biomarker identification, drug development, and immunotherapies.
Modern MS-based proteomics provides an opportunity to systematically characterize proteins. However, due to the complexity of cell-surface proteins, the labor-intensive workflow, and the limit of clinical samples, comprehensive characterization of the surfaceome remains extraordinarily challenging, especially in clinical studies. Developing and optimizing surfaceome enrichment methods and utilizing automated sample preparation workflow can expand the applications of surfaceome analysis and deepen our understanding of the functions of cell-surface proteins.
The cell surface contains many important proteins such as receptors and transporters. These proteins are responsible for cells to communicate with each other, take nutrients from outside, and interact with their surroundings. Aberrant changes in surface protein expression, modifications, and interactions with other molecules directly result in various diseases, including infections, immune disorders, and cancer. Currently, mass spectrometry (MS)-based proteomics is very powerful to study proteins on a large scale, and there has been a strong interest in employing MS to investigate cell-surface proteins. In this review, we discuss different methods combining mass spectrometry with other approaches to systematically characterize protein abundance, dynamics, modification, and interaction on the cell surface. These methods help uncover protein functions and specific cell-surface proteins related to human diseases. A better understanding of the functions and properties of cell-surface proteins can facilitate the discovery of surface proteins as effective biomarkers for disease early detection and the identification of drug targets for disease treatment.

Colorectal cancer (CRC) ranks as the third most prevalent cancer globally, and about half of CRC patients eventually succumb to tumor metastasis. Despite this, treatment options for metastatic colon cancer remain severely limited, reflected by a 12% 5-year overall survival rate. Increasing evidence suggests that cancer stem cells (CSCs) are pivotal in driving CRC metastasis. Our study found a significant upregulation of MOGS in metastatic colorectal cancer, with high MOGS expression inversely correlating with patient prognosis. Additionally, MOGS enhances the NOTCH pathway, thus promoting stemness in CRC cells, both in vitro and in vivo. Mechanistically, MOGS may facilitate the maturation of NOTCH1 protein by promoting NOTCH1 glycosylation. Correspondingly, silencing MOGS markedly reduced invasion and stemness of CRC cells in vivo. In summary, our findings highlight the critical role of MOGS in fostering stemness and activating the NOTCH pathway in colorectal cancer cells. Disrupting the function of the MOGS/NOTCH could represent a feasible therapeutic strategy for CRC management.

Parkinson\'s disease (PD) associated state of neuroinflammation due to the aggregation of aberrant proteins is widely reported. One type of post-translational modification involved in protein stability is glycosylation. Here, we aimed to characterize the human Parkinsonian nigro-striatal N-glycome, and related transcriptome/proteome, and its correlation with endoplasmic reticulum (ER) stress and unfolded protein response (UPR), providing a comprehensive characterization of the PD molecular signature. Significant changes were seen upon a PD: a 3% increase in sialylation and 5% increase in fucosylation in both regions, and a 2% increase in oligomannosylated N-glycans in the substantia nigra. In the latter, a decrease in the mRNA expression of sialidases and an upregulation in the UPR pathway were also seen. To show the correlation between these, we also describe a small in vitro study where changes in specific glycosylation trait enzymes (inhibition of sialyltransferases) led to impairments in cell mitochondrial activity, changes in glyco-profile, and upregulation in UPR pathways. This complete characterization of the human nigro-striatal N-glycome provides an insight into the glycomic profile of PD through a transversal approach while combining the other PD \"omics\" pieces, which can potentially assist in the development of glyco-focused therapeutics.

Streptococcus mutans is commonly associated with dental caries and the ability to form biofilms is essential for its pathogenicity. We recently identified the Pgf glycosylation machinery of S. mutans, responsible for the post-translational modification of the surface-associated adhesins Cnm and WapA. Since the four-gene pgf operon (pgfS-pgfM1-pgfE-pgfM2) is part of the S. mutans core genome, we hypothesized that the scope of the Pgf system goes beyond Cnm and WapA glycosylation. In silico analyses and tunicamycin sensitivity assays suggested a functional overlap between the Pgf machinery and the rhamnose-glucose polysaccharide synthesis pathway. Phenotypic characterization of pgf mutants (ΔpgfS, ΔpgfE, ΔpgfM1, ΔpgfM2, and Δpgf) revealed that the Pgf system is important for biofilm formation, surface charge, membrane stability, and survival in human saliva. Moreover, deletion of the entire pgf operon (Δpgf strain) resulted in significantly impaired colonization in a rat oral colonization model. Using Cnm as a model, we showed that Cnm is heavily modified with N-acetyl hexosamines but it becomes heavily phosphorylated with the inactivation of the PgfS glycosyltransferase, suggesting a crosstalk between these two post-translational modification mechanisms. Our results revealed that the Pgf machinery contributes to multiple aspects of S. mutans pathobiology that may go beyond Cnm and WapA glycosylation.

Unlike glycosylation of proteins expressed in mammalian systems, bacterial glycosylation is often neglected in the development of recombinant vaccines.
Here, we compared the effects of glycosylation of YghJ, an Escherichia coli protein important for mucus attachment of bacteria causing in urinary tract infections (UTIs). A novel method based on statistical evaluation of phage display for the identification and comparison of epitopes and mimotopes of anti-YghJ antibodies in the sera was used. This is the first time that the effect of glycosylation of a recombinant bacterial antigen has been studied at the peptide epitope level.
The study identifies differences in the immune response for (non)-glycosylated antigens in rabbits and pigs and compares them to a large group of patients with UTI, which have been diagnosed as positive for various bacterial pathogens. We identified glycosylation-specific peptide epitopes, a large immunological similarity between different UTI pathogens, and a broad peptide epitope pattern in patients and animals, which could result in a variable response in patients upon vaccination.
This epitope analysis indicates that the vaccination of rabbits and pigs raises antibodies that translate well into the human immune system. This study underlines the importance of glycosylation in bacterial vaccines and provides detailed immune diagnostic methods to understand individual immune responses to vaccines.

Eukaryotic dehydrodolichyl diphosphate synthases (DHDDSs), cis-prenyltransferases (cis-PTs) synthesizing precursors of dolichols to mediate glycoprotein biosynthesis require partners, for eample Nus1 in yeast and NgBR in animals, which are cis-PTs homologues without activity but to boost the DHDDSs activity. Unlike animals, plants have multiple cis-PT homologues to pair or stand alone to produce various chain-length products with less known physiological roles. We chose Cinnamomum kanehirae, a tree that contains two DHDDS-like and three NgBR-like proteins from genome analysis, and found that one DHDDS-like protein acted as a homodimeric cis-PT to make a medium-chain C55 product, while the other formed heterodimeric complexes with either one of two NgBR homologues to produce longer-chain products. Both complexes were functional to complement the growth defect of the yeast rer2 deficient strain at a higher temperature. From the roles for the polyprenol and dolichol biosynthesis and sequence motifs, their homologues in various species were compared to reveal their possible evolutionary paths.

UNASSIGNED: Glycans attached to immunoglobulin G are an important regulator of chronic systemic inflammation, one of the key drivers of aging. As people age, glycans that suppress inflammation are being replaced with inflammation-promoting glycans, but the rate of this conversion is highly individual and is affected by genetic, epigenetic, and environmental factors.
UNASSIGNED: This review summarizes key studies of IgG glycosylation changes in aging and disease, effects of lifestyle and pharmacological interventions, and mechanisms that regulate IgG glycosylation.
UNASSIGNED: IgG glycome is an important contributor to the process of aging that can be modulated by both lifestyle and pharmacological interventions. Small molecule drugs that would suppress chronic systemic inflammation by modulation of the IgG glycome are still not available, but since gene network regulating IgG glycosylation has been identified and a high-throughput in vitro screening system is available, it is likely that this highly innovative approach to manage chronic systemic inflammation will be developed soon.

Breast cancer represents a paramount global health challenge, warranting intensified exploration of the molecular underpinnings influencing its progression to facilitate the development of precise diagnostic instruments and customized therapeutic regimens. Historically, the Golgi apparatus has been acknowledged for its primary role in protein sorting and trafficking within cellular contexts. However, recent findings suggest a potential link between modifications in Golgi apparatus function and organization and the pathogenesis of breast cancer. This review delivers an exhaustive analysis of this correlation. Specifically, we examine the consequences of disrupted protein glycosylation, compromised protein transport, and inappropriate oncoprotein processing on breast cancer cell dynamics. Furthermore, we delve into the impacts of Golgi-mediated secretory routes on the release of pro-tumorigenic factors during the course of breast cancer evolution. Elucidating the nuanced interplay between the Golgi apparatus and breast cancer can pave the way for innovative therapeutic interventions and the discovery of biomarkers, potentially enhancing the diagnostic, prognostic, and therapeutic paradigms for afflicted patients. The advancement of such research could substantially expedite the realization of these objectives.