glycomics-guided-glycoproteomics-facilitates-comprehensive-profiling

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments 

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

Macquarie University

N-Glycoproteomics, the system-wide study of glycans asparagine-linked to protein carriers, holds a unique and still largely untapped potential to provide deep insights into the complexity and dynamics of the heterogeneous N-glycoproteome. Despite the advent of innovative analytical and informatics tools aiding the analysis, N-glycoproteomics remains challenging and consequently largely restricted to specialised laboratories. Aiming to stimulate discussions of method harmonisation, data standardisation and reporting guidelines to make N-glycoproteomics more reproducible and accessible to the community, we here discuss critical considerations related to the design and execution of N-glycoproteomics experiments and highlight good practices in N-glycopeptide data collection, analysis, interpretation and sharing. Giving the rapid maturation and, expectedly, a wide-spread implementation of N-glycoproteomics capabilities across the community in future years, this piece aims to point out common pitfalls, to encourage good data sharing and documentation practices, and to highlight practical solutions and strategies to enhance the insight into the N-glycoproteome.

Critical considerations in N-glycoproteomics.

Mass spectrometry-driven glycomics and glycoproteomics, the system-wide profiling of detached glycans and intact glycopeptides from biological samples, respectively, are powerful approaches to interrogate the heterogenous glycoproteome. Efforts to develop integrated workflows employing both glycomics and glycoproteomics have been invested since the concerted application of these complementary approaches enables a deeper exploration of the glycoproteome. This protocol paper outlines, step-by-step, an integrated -omics technology, the "glycomics-assisted glycoproteomics" method, that first establishes the N-glycan fine structures and their quantitative distribution pattern of protein extracts via porous graphitized carbon-LC-MS/MS. The N-glycome information is then used to augment and guide the challenging reversed-phase LC-MS/MS-based profiling of intact N-glycopeptides from the same protein samples. Experimental details and considerations relating to the sample preparation and the N-glycomics and N-glycoproteomics data collection, analysis, and integration are discussed. Benefits of the glycomics-assisted glycoproteomics method, which can be readily applied to both simple and complex biological specimens such as protein extracts from cells, tissues, and bodily fluids (e.g., serum), include quantitative information of the protein carriers and site(s) of glycosylation, site occupancy, and the site-specific glycan structures directly from biological samples. The glycomics-assisted glycoproteomics method therefore facilitates a comprehensive view of the complexity and dynamics of the heterogenous glycoproteome.

Glycomics-Assisted Glycoproteomics Enables Deep and Unbiased N-Glycoproteome Profiling of Complex Biological Specimens.

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets' tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.

Analysis of the Healthy Platelet Proteome Identifies a New Form of Domain-Specific O-Fucosylation.

The morbidity and mortality of sepsis remain high. Clinicians lack effective markers to rapidly diagnose sepsis and identify the underlying pathogen infection particularly for patients with candidaemia or cases of culture-negative sepsis where culture-based diagnostics are inadequate. In our search for new lines of potential sepsis biomarkers, we here explore the impact of various classes of infectious agents on the serum -glycome in a septic shock cohort. Comparative -glycomics was performed on sera collected from 49 septic shock patients infected with viral (n = 9), bacterial (n = 37) or fungal (n = 3) pathogens using an established PGC-LC-MS/MS method. Aberrant serum -glycosylation features were observed in patients with fungal infection relative to the other infection sub-groups including i) altered expression of prominent α2,6-sialylated biantennary -glycan isomers, ii) elevated levels of IgG-type -glycosylation and iii) a global shift in the serum -glycome involving altered glycan type distribution and considerable changes in core fucosylation and α2,6-sialylation. Septic shock patients infected with bacterial and viral pathogens exhibited similar global serum -glycome features and therefore could not be stratified based on their serum -glycosylation. Subtle and less consistent serum -glycome differences were observed between septic shock patients infected with different bacterial pathogens. In conclusion, our study has tested the impact of different pathogen classes on the serum -glycome in a septic shock cohort, and reports that fungal infection impacts the host serum -glycome differently compared to bacterial or viral infections thus potentially opening avenues for glycan-based biomarkers to better diagnose patients with candidaemia.

The Huong Chau

School of Natural Sciences,
Faculty of Science and Engineering,
School of Natural Sciences, Faculty of Science and Engineering

Macquarie University

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

The Huong Chau

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Natural Sciences,Faculty of Science and Engineering,School of Natural Sciences, Faculty of Science and Engineering

Macquarie University

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

School of Natural Sciences,
Faculty of Science and Engineering,
School of Natural Sciences, Faculty of Science and Engineering