scholarly journals Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis

2019 ◽  
Author(s):  
Nicholas M. Riley ◽  
Alexander S. Hebert ◽  
Michael S. Westphall ◽  
Joshua J. Coon
Keyword(s):  
Large Scale ◽  
Electron Transfer Dissociation ◽  
Protein Glycosylation ◽  
Analytical Challenge ◽  
Post Translational Modification ◽  
Structural Domains ◽  
Site Specific ◽  
Future Studies ◽  
Site Heterogeneity ◽  
Degree Of Heterogeneity

ABSTRACTProtein glycosylation is a highly important, yet a poorly understood protein post-translational modification. Thousands of possible glycan structures and compositions create potential for tremendous site heterogeneity and analytical challenge. A lack of suitable analytical methods for large-scale analyses of intact glycopeptides has ultimately limited our abilities to both address the degree of heterogeneity across the glycoproteome and to understand how it contributes biologically to complex systems. Here we show that N-glycoproteome site-specific microheterogeneity can be captured via large-scale glycopeptide profiling with methods enabled by activated ion electron transfer dissociation (AI-ETD), ultimately characterizing 1,545 N-glycosites (>5,600 unique N-glycopeptides) from mouse brain tissue. Moreover, we have used this large-scale glycoproteomic data to develop several new visualizations that will prove useful for analyzing intact glycopeptides in future studies. Our data reveal that N-glycosylation profiles can differ between subcellular regions and structural domains and that N-glycosite heterogeneity manifests in several different forms, including dramatic differences in glycosites on the same protein.

scholarly journals Optimal Dissociation Methods Differ for N- and O-glycopeptides

2020 ◽  
Author(s):  
Nicholas Riley ◽  
Stacy A. Malaker ◽  
Marc D. Driessen ◽  
Carolyn Bertozzi
Keyword(s):  
Large Scale ◽  
Collision Energy ◽  
Electron Transfer Dissociation ◽  
Method Performance ◽  
Site Specific ◽  
Advantages And Disadvantages ◽  
Beam Type ◽  
Supplemental Activation ◽  
Glycopeptide Analysis

<p><a>Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.</a></p>

scholarly journals SugarPy facilitates the universal, discovery-driven analysis of intact glycopeptides

2020 ◽  
Author(s):  
Stefan Schulze ◽  
Anne Oltmanns ◽  
Christian Fufezan ◽  
Julia Krägenbring ◽  
Michael Mormann ◽  
...  
Keyword(s):  
Large Scale ◽  
Protein Glycosylation ◽  
Supplementary Information ◽  
Cyanidioschyzon Merolae ◽  
Human Breast Milk ◽  
The Novel ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Domains Of Life

AbstractMotivationProtein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes.ResultsTherefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii. SugarPy also facilitated the novel characterization of glycoproteins from the red alga Cyanidioschyzon merolae.AvailabilityThe source code is freely available on GitHub (https://github.com/SugarPy/SugarPy), and its implementation in Python ensures support for all operating [email protected] and [email protected] informationSupplementary data are available online.

scholarly journals Glycans as Biomarkers: Status and Perspectives

2011 ◽  
Vol 30 (3) ◽  
pp. 213-223 ◽  
Author(s):  
Miroslava Janković
Keyword(s):  
Large Scale ◽  
Biomarker Discovery ◽  
High Sensitivity ◽  
Disease Diagnosis ◽  
Protein Glycosylation ◽  
Post Translational Modification ◽  
Different Types ◽  
Progression Of Disease ◽  
New Biomarkers ◽  
Selection Of

Glycans as Biomarkers: Status and PerspectivesProtein glycosylation is a ubiquitous and complex co- and post-translational modification leading to glycan formation, i.e. oligosaccharide chains covalently attached to peptide backbones. The significance of changes in glycosylation for the beginning, progress and outcome of different human diseases is widely recognized. Thus, glycans are considered as unique structures to diagnose, predict susceptibility to and monitor the progression of disease. In the »omics« era, the glycome, a glycan analogue of the proteome and genome, holds considerable promise as a source of new biomarkers. In the design of a strategy for biomarker discovery, new principles and platforms for the analysis of relatively small amounts of numerous glycoproteins are needed. Emerging glycomics technologies comprising different types of mass spectrometry and affinity-based arrays are next in line to deliver new analytical procedures in the field of biomarkers. Screening different types of glycomolecules, selection of differentially expressed components, their enrichment and purification or identification are the most challenging parts of experimental and clinical glycoproteomics. This requires large-scale technologies enabling high sensitivity, proper standardization and validation of the methods to be used. Further progress in the field of applied glycoscience requires an integrated systematic approach in order to explore properly all opportunities for disease diagnosis.

scholarly journals Mapping In Vivo O-Glycoproteome Using Site-specific Extraction of O-linked glycopeptides (EXoO)

2018 ◽  
Author(s):  
Weiming Yang ◽  
Minghui Ao ◽  
Yingwei Hu ◽  
Qing Kay Li ◽  
Hui Zhang
Keyword(s):  
Large Scale ◽  
Human Kidney ◽  
Clinical Diagnostics ◽  
Protein Glycosylation ◽  
Major Type ◽  
Site Specific ◽  
Detailed Structural Analysis ◽  
Glycosylation Sites ◽  
Definition Of

AbstractProtein glycosylation is one of the most abundant post-translational modifications. However, detailed analysis ofin vivoO-linked glycosylation, a major type of protein glycosylation, has been severely impeded by the scarcity of suitable methodologies. Here, we present a chemoenzymatic method for the site-specificextractionofO-linked glycopeptides (EXoO), which enabled the unambiguous mapping of over 3,000 O-linked glycosylation sites and definition of their glycans on over 1,000 proteins in human kidney tissues, T cells and serum. This large-scale localization of O-linked glycosylation sites nearly doubles the number of previously identified sites, demonstrating that EXoO is the most effective method to-date for defining the site-specific O-linked glycoproteome in different types of sample. Detailed structural analysis of the sites identified revealed conserved motifs and topological orientations facing extracellular space, the cell surface, the lumen of the ER and the Golgi. EXoO was also able to reveal significant differences in thein vivoO-linked glycoproteome of tumor and normal kidney tissues pointing to its broader use in clinical diagnostics and therapeutics.

scholarly journals Optimal Dissociation Methods Differ for N- and O-glycopeptides

2020 ◽  
Author(s):  
Nicholas Riley ◽  
Stacy A. Malaker ◽  
Marc D. Driessen ◽  
Carolyn Bertozzi
Keyword(s):  
Large Scale ◽  
Collision Energy ◽  
Electron Transfer Dissociation ◽  
Method Performance ◽  
Site Specific ◽  
Advantages And Disadvantages ◽  
Beam Type ◽  
Supplemental Activation ◽  
Glycopeptide Analysis

<p><a>Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.</a></p>

scholarly journals SugarPy facilitates the universal, discovery-driven analysis of intact glycopeptides

2020 ◽  
Author(s):  
Stefan Schulze ◽  
Anne Oltmanns ◽  
Christian Fufezan ◽  
Julia Krägenbring ◽  
Michael Mormann ◽  
...  
Keyword(s):  
Large Scale ◽  
Protein Glycosylation ◽  
Supplementary Information ◽  
Cyanidioschyzon Merolae ◽  
Human Breast Milk ◽  
The Novel ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Domains Of Life

Abstract Motivation Protein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes. Results Therefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii. SugarPy also facilitated the novel characterization of glycoproteins from the red alga Cyanidioschyzon merolae. Availability and implementation The source code is freely available on GitHub (https://github.com/SugarPy/SugarPy), and its implementation in Python ensures support for all operating systems. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Optimal Dissociation Methods Differ for N- and O-glycopeptides

2020 ◽  
Author(s):  
Nicholas Riley ◽  
Stacy A. Malaker ◽  
Marc D. Driessen ◽  
Carolyn Bertozzi
Keyword(s):  
Large Scale ◽  
Collision Energy ◽  
Electron Transfer Dissociation ◽  
Method Performance ◽  
Site Specific ◽  
Advantages And Disadvantages ◽  
Beam Type ◽  
Supplemental Activation ◽  
Glycopeptide Analysis

<p><a>Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.</a></p>

scholarly journals Reanalysis of an Allocentric Navigation Strategy Scale based on Item Response Theory

2020 ◽  
Vol 2 (1) ◽  
pp. 90-105
Author(s):  
Jimmy Y. Zhong
Keyword(s):  
Item Response Theory ◽  
Item Response ◽  
Large Scale ◽  
Relative Magnitude ◽  
Latent Structure ◽  
Response Theory ◽  
Future Studies ◽  
Bidimensional Model ◽  
Navigation Strategy ◽  
Component Loading

AbstractFocusing on 12 allocentric/survey-based strategy items of the Navigation Strategy Questionnaire (Zhong & Kozhevnikov, 2016), the current study applied item response theory-based analysis to determine whether a bidimensional model could better describe the latent structure of the survey-based strategy. Results from item and model fit diagnostics, categorical response and item information curves showed that an item with the lowest rotated component loading (.27) [SURVEY12], could be considered for exclusion in future studies; and that a bidimensional model with three preference-related items constituting a content factor offered a better representation of the latent structure than a unidimensional model per se. Mean scores from these three items also correlated significantly with a pointing-to-landmarks task to the same relative magnitude as the mean scores from all items, and all items excluding SURVEY12. These findings gave early evidence suggesting that the three preference-related items could constitute a subscale for deriving quick estimates of large-scale allocentric spatial processing in healthy adults in both experimental and clinical settings. Potential cognitive and brain mechanisms were discussed, followed by calls for future studies to gather greater evidence confirming the predictive validity of the full and sub scales, along with the design of new items focusing on environmental familiarity.

scholarly journals Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander Pralow ◽  
Alexander Nikolay ◽  
Arnaud Leon ◽  
Yvonne Genzel ◽  
Erdmann Rapp ◽  
...  
Keyword(s):  
Zika Virus ◽  
Animal Cell ◽  
Gradient Centrifugation ◽  
Viral Envelope ◽  
Protein Glycosylation ◽  
High Yield ◽  
Structural Protein ◽  
Site Specific ◽  
E Protein ◽  
The Impact

AbstractHere, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC–MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

scholarly journals Long-term effectiveness of faecal immunochemical test screening for proximal and distal colorectal cancers

Gut ◽  
2021 ◽  
pp. gutjnl-2020-322545
Author(s):  
Han-Mo Chiu ◽  
Grace Hsiao-Hsuan Jen ◽  
Ying-Wei Wang ◽  
Jean Ching-Yuan Fann ◽  
Chen-Yang Hsu ◽  
...  
Keyword(s):  
Large Scale ◽  
Distal Colon ◽  
Population Based ◽  
Proximal Colon ◽  
Advanced Stage ◽  
Site Specific ◽  
Faecal Immunochemical Test ◽  
Immunochemical Test ◽  
Selection Factors

ObjectiveTo measure the effects of faecal immunochemical test (FIT) for colorectal cancer (CRC) screening on overall and site-specific long-term effectiveness of population-based organised service screening.DesignA prospective cohort study of Taiwanese nationwide biennial FIT screening was performed. A total of 5 417 699 eligible subjects were invited to attend screening from 2004 through 2009 and were followed up until 2014. We estimated the adjusted relative rates (aRRs) on the effectiveness of reducing advanced-stage CRC (stage II+) and CRC death by Bayesian Poisson regression models with the full adjustment for a cascade of self-selection factors (including the screening rate and the colonoscopy rate) and the completeness of colonoscopy together with demographic features.ResultsFIT screening (exposed vs unexposed) reduced the incidence of advanced-stage CRC (48.4 vs 75.7 per 100 000) and mortality (20.3 vs 41.3 per 100 000). Statistically significant reductions of both incidence of advanced-stage CRCs (aRR=0.66, 95% CI 0.63 to 0.70) and deaths from CRC (aRR=0.60, 95% CI 0.57 to 0.64) were noted. FIT screening was more effective in reducing distal advanced-stage CRCs (aRR=0.61, 95% CI 0.58 to 0.64) and CRC mortality (aRR=0.56, 95% CI 0.53 to 0.69) than proximal advanced CRCs (aRR=0.84, 95% CI 0.77 to 0.92) and CRC mortality (aRR=0.72, 95% CI 0.66 to 0.80).ConclusionA large-scale population-based biennial FIT screening demonstrates 34% significant reduction of advanced-stage CRCs and 40% reduction of death from CRC with larger long-term effectiveness in the distal colon than the proximal colon. Our findings provide a strong and consistent evidence-based policy for supporting a sustainable population-based FIT organised service screening worldwide. The disparity of site-specific long-term effectiveness also provides an insight into the remedy for lower effectiveness of FIT screening in the proximal colon.

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