Integrated serum proteomic and N-glycoproteomic characterization of dengue patients.

Dengue fever is a mosquito-borne viral disease caused by the dengue virus (DENV). It poses a public health threat globally and, while most people with dengue have mild symptoms or are asymptomatic, approximately 5% of affected individuals develop severe disease and need hospital care. However, knowledge of the molecular mechanisms underlying dengue infection and the interaction between the virus and its host remains limited. In the present study, we performed a quantitative proteomic and N-glycoproteomic analysis of serum from 19 patients with dengue and 11 healthy people. The results revealed distinct proteomic and N-glycoproteomic landscapes between the two groups. Notably, we report for the first time the changes in the serum N glycosylation pattern following dengue infection and provide abundant information on glycoproteins, glycosylation sites, and intact N-glycopeptides using recently developed site-specific glycoproteomic approaches. Furthermore, a series of key functional pathways in proteomic and N-glycoproteomic were identified. Collectively, our findings significantly improve understanding of host and DENV interactions and the general pathogenesis and pathology of DENV, laying a foundation for functional studies of glycosylation and glycan structures in dengue infection.

Glycoproteomic Analysis of Urinary Extracellular Vesicles for Biomarkers of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) accounts for the most common form of primary liver cancer cases and constitutes a major health problem worldwide. The diagnosis of HCC is still challenging due to the low sensitivity and specificity of the serum α-fetoprotein (AFP) diagnostic method. Extracellular vesicles (EVs) are heterogeneous populations of phospholipid bilayer-enclosed vesicles that can be found in many biological fluids, and have great potential as circulating biomarkers for biomarker discovery and disease diagnosis. Protein glycosylation plays crucial roles in many biological processes and aberrant glycosylation is a hallmark of cancer. Herein, we performed a comprehensive glycoproteomic profiling of urinary EVs at the intact N-glycopeptide level to screen potential biomarkers for the diagnosis of HCC. With the control of the spectrum-level false discovery rate ≤1%, 756 intact N-glycopeptides with 154 N-glycosites, 158 peptide backbones, and 107 N-glycoproteins were identified. Out of 756 intact N-glycopeptides, 344 differentially expressed intact N-glycopeptides (DEGPs) were identified, corresponding to 308 upregulated and 36 downregulated N-glycopeptides, respectively. Compared to normal control (NC), the glycoproteins LG3BP, PIGR and KNG1 are upregulated in HCC-derived EVs, while ASPP2 is downregulated. The findings demonstrated that specific site-specific glycoforms in these glycoproteins from urinary EVs could be potential and efficient non-invasive candidate biomarkers for HCC diagnosis.

Mesoporous covalent organic framework microspheres with dual-phase separation strategy for high-purity glycopeptide enrichment.

High-performance porous materials and rational enrichment strategies are crucial during sample pretreatment process in glycoproteomics analysis. Herein, we report a dual-phase separation strategy based on hydrophobic and hydrophilic mesoporous covalent-organic framework (COF) microspheres for high-purity glycopeptide enrichment for the first time. The COF microspheres (about 1.8±0.5 µm) with hydrophobic mesopores (2.5 nm) were prepared by a facile method at room temperature. Through the post-synthesis modification strategy, hydrophilic mesopores were obtained by modifying the vinyl ligands with glutathione (GSH), and the hydrophilic properties of COF microspheres were further enhanced by the introduction of Au nanoparticles and GSH to obtain the hydrophilic COF microspheres (denoted as COF@Au-GSH). The low-abundance hydrophilic glycopeptides could be efficiently enriched by the hydrophilic COF@Au-GSH microspheres in low polar solutions after the high-abundance hydrophobic non-glycopeptides were removed with the hydrophobic COF microspheres in high polar solutions. With the help of dual-phase separation strategy and inherent properties of the COF structure, the as-prepared COF microspheres showed splendid enrichment performance for glycopeptides, including ultrahigh sensitivity (2 fmol, IgG digests), extremely high specificity (1:10000, IgG digests/BSA digests), excellent size selectivity (1:500:500, IgG digests/BSA/IgG), and large binding capacity (200 mg g-1, IgG digests). In addition, a total of 1993 glycopeptides could be enriched and identified from the rat liver digests after enrichment by the COF microspheres. As a proof of concept application, the proposed strategy was successfully used in sample pretreatment process for plasma glycoproteomic analysis.

Serum Glycoprotein Markers in Nonalcoholic Steatohepatitis and Hepatocellular Carcinoma.

Fatty liver disease progresses through stages of fat accumulation and inflammation to nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis, and eventually hepatocellular carcinoma (HCC). Currently available diagnostic tools for HCC lack sensitivity and specificity. In this study, we investigated the use of circulating serum glycoproteins to identify a panel of potential prognostic markers that may be indicative of progression from the healthy state to NASH and further to HCC. Serum samples were processed and analyzed using a novel high-throughput glycoproteomics platform. Our initial dataset contained healthy, NASH, and HCC serum samples. We analyzed 413 glycopeptides, representing 57 abundant serum proteins, and compared among the three phenotypes. We studied the normalized abundance of common glycoforms and found 40 glycopeptides with statistically significant differences in abundances in NASH and HCC compared to controls. Summary level relative abundances of core-fucosylated, sialylated, and branched glycans containing glycopeptides were higher in NASH and HCC as compared to controls. We replicated some of our findings in an independent set of samples of individuals with benign liver conditions and HCC. Our results may be of value in the management of liver diseases. Data generated in this work can be downloaded from MassIVE (https://massive.ucsd.edu) with identifier MSV000088809.

Semi-Automated Glycoproteomic Data Analysis of LC-MS Data Using GlycopeptideGraphMS in Process Development of Monoclonal Antibody Biologics.

The glycosylation of antibody-based proteins is vital in translating the right therapeutic outcomes of the patient. Despite this, significant infrastructure is required to analyse biologic glycosylation in various unit operations from biologic development, process development to QA/QC in bio-manufacturing. Simplified mass spectrometers offer ease of operation as well as the portability of method development across various operations. Furthermore, data analysis would need to have a degree of automation to relay information back to the manufacturing line. We set out to investigate the applicability of using a semiautomated data analysis workflow to investigate glycosylation in different biologic development test cases. The workflow involves data acquisition using a BioAccord LC-MS system with a data-analytical tool called GlycopeptideGraphMS along with Progenesis QI to semi-automate glycoproteomic characterisation and quantitation with a LC-MS1 dataset of a glycopeptides and peptides. Data analysis which involved identifying glycopeptides and their quantitative glycosylation was performed in 30 min with minimal user intervention. To demonstrate the effectiveness of the antibody and biologic glycopeptide assignment in various scenarios akin to biologic development activities, we demonstrate the effectiveness in the filtering of IgG1 and IgG2 subclasses from human serum IgG as well as innovator drugs trastuzumab and adalimumab and glycoforms by virtue of their glycosylation pattern. We demonstrate a high correlation between conventional released glycan analysis with fluorescent tagging and glycopeptide assignment derived from GraphMS. GraphMS workflow was then used to monitor the glycoform of our in-house trastuzumab biosimilar produced in fed-batch cultures. The demonstrated utility of GraphMS to semi-automate quantitation and qualitative identification of glycopeptides proves to be an easy data analysis method that can complement emerging multi-attribute monitoring (MAM) analytical toolsets in bioprocess environments.

Potential Plasticity of the Mannoprotein Repertoire Associated to Mycobacterium tuberculosis Virulence Unveiled by Mass Spectrometry-Based Glycoproteomics.

To date, Mycobacterium tuberculosis (Mtb) remains the world's greatest infectious killer. The rise of multidrug-resistant strains stresses the need to identify new therapeutic targets to fight the epidemic. We previously demonstrated that bacterial protein-O-mannosylation is crucial for Mtb infectiousness, renewing the interest of the bacterial-secreted mannoproteins as potential drug-targetable virulence factors. The difficulty of inventorying the mannoprotein repertoire expressed by Mtb led us to design a stringent multi-step workflow for the reliable identification of glycosylated peptides by large-scale mass spectrometry-based proteomics. Applied to the differential analyses of glycoproteins secreted by the wild-type Mtb strain-and by its derived mutant invalidated for the protein-O-mannosylating enzyme PMTub-this approach led to the identification of not only most already known mannoproteins, but also of yet-unknown mannosylated proteins. In addition, analysis of the glycoproteome expressed by the isogenic recombinant Mtb strain overexpressing the PMTub gene revealed an unexpected mannosylation of proteins, with predicted or demonstrated functions in Mtb growth and interaction with the host cell. Since in parallel, a transient increased expression of the PMTub gene has been observed in the wild-type bacilli when infecting macrophages, our results strongly suggest that the Mtb mannoproteome may undergo adaptive regulation during infection of the host cells. Overall, our results provide deeper insights into the complexity of the repertoire of mannosylated proteins expressed by Mtb, and open the way to novel opportunities to search for still-unexploited potential therapeutic targets.

Glycoproteoform Profiles of Individual Patients' Plasma Alpha-1-Antichymotrypsin are Unique and Extensively Remodeled Following a Septic Episode.

Sepsis and septic shock remain the leading causes of death in intensive care units (ICUs), yet the pathogenesis originating from the inflammatory response during sepsis remains ambiguous. Acute-phase proteins are typically highly glycosylated, and the nature of the glycans have been linked to the incidence and severity of such inflammatory responses. To further build upon these findings we here monitored, the longitudinal changes in the plasma proteome and, in molecular detail, glycoproteoform profiles of alpha-1-antichymotrypsin (AACT) extracted from plasma of ten individual septic patients. For each patient we included four different time-points, including post-operative (before sepsis) and following discharge from the ICU. We isolated AACT from plasma depleted for albumin, IgG and serotransferrin and used high-resolution native mass spectrometry to qualitatively and quantitatively monitor the multifaceted glycan microheterogeneity of desialylated AACT, which allowed us to monitor how changes in the glycoproteoform profiles reflected the patient's physiological state. Although we observed a general trend in the remodeling of the AACT glycoproteoform profiles, e.g. increased fucosylation and branching/LacNAc elongation, each patient exhibited unique features and responses, providing a resilient proof-of-concept for the importance of personalized longitudinal glycoproteoform profiling. Importantly, we observed that the AACT glycoproteoform changes induced by sepsis did not readily subside after discharge from ICU.

Development of a glycoproteomic strategy to detect more aggressive prostate cancer using lectin-immunoassays for serum fucosylated PSA.

BACKGROUND: Prostate-specific antigen (PSA) is commonly used as a serum biomarker for the detection of prostate cancer. However, levels of PSA in serum do not reliably distinguish aggressive prostate cancer from non-aggressive disease. Therefore, there is an urgent need for biomarkers that can differentiate aggressive prostate cancers from non-aggressive phenotypes. Fucosylation is one of the glycosylation-based protein modifications. Previously we demonstrated increased levels of serum fucosylated PSA in patients with aggressive prostate cancer using lectin selection followed by PSA immunoassay. METHODS: We developed two lectin-immunoassays, Lens culinaris agglutinin (LCA) and Aleuria aurantia lectin (AAL) followed by clinical PSA immunoassay and investigated the levels of PSA and its fucosylated glycoforms in serum specimens from prostate cancer patients with different Gleason scores. First, we developed standard curves for lectins enrichment, which were applied to lectin-immunoassay for fucosylated PSA-LCA and PSA-AAL quantification in serum samples. RESULTS: Our results showed that both LCA- and AAL-immunoassays detected elevated fucosylated PSA and were correlated with higher Gleason scores but only AAL-immunoassay detected an increased percentage of fucosylated PSA in patient serum with higher Gleason scores. CONCLUSION: We have developed quantitative lectin-immunoassays for serum fucosylated PSA. Our data demonstrated that fucosylated PSA-AAL, % fucosylated PSA-AAL and fucosylated PSA-LCA levels could be effective biomarkers to differentiate aggressive prostate cancer [especially Gleason 7 (4 + 3) or above] from non-aggressive disease. We believe that application of these lectin-immunoassays to a larger patient population is needed to evaluate the clinical utilities of fucosylated PSA using AAL-PSA and LCA-PSA for aggressive prostate cancer.

Proteomic and N-glycoproteomic quantification reveal aberrant changes in the human saliva of oral ulcer patients.

Human whole saliva is a vital body fluid for studying the physiology and pathology of the oral cavity. As a powerful technique for biomarker discovery, MS-based proteomic strategies have been introduced for saliva analysis and identified hundreds of proteins and N-glycosylation sites. However, there is still a lack of quantitative analysis, which is necessary for biomarker screening and biological research. In this study, we establish an integrated workflow by the combination of stable isotope dimethyl labeling, HILIC enrichment, and high resolution MS for both quantification of the global proteome and N-glycoproteome of human saliva from oral ulcer patients. With the help of advanced bioinformatics, we comprehensively studied oral ulcers at both protein and glycoprotein scales. Bioinformatics analyses revealed that starch digestion and protein degradation activities are inhibited while the immune response is promoted in oral ulcer saliva.

Comparative secretomic and N-glycoproteomic profiling in human MCF-7 breast cancer and HMEpC normal epithelial cell lines using a gel-based strategy.

BACKGROUND: Concurrent study of secretomic and glycoproteomic profiles in cancer cell lines represents an excellent approach for investigating cancer progression and identifying novel biomarker candidates. In this study, we performed a comparative secretomic and N-glycoprotein profiling from the secretions of normal human mammary epithelial cells (HMEpC) and the MCF-7 human breast cancer cell line. METHOD: We analyzed these cell lines using a combined methodology involving glycan-binding lectins and two-dimensional electrophoresis and identified several differentially secreted factors, including osteonectin and haptoglobin. RESULT: Notably, comparative analyses also revealed that MCF-7 cells produced differentially N-glycosylated forms of haptoglobin. CONCLUSION: The present data suggested that osteonectin and haptoglobin might have potential to be served as potential biomarkers for breast cancer. However, further investigation is needed to validate the findings.