Site-specific protein glycosylation analysis with glycan isomer differentiation

2011 ◽  
Vol 403 (5) ◽  
pp. 1291-1302 ◽  
Author(s):  
Serenus Hua ◽  
Charles C. Nwosu ◽  
John S. Strum ◽  
Richard R. Seipert ◽  
Hyun Joo An ◽  
...  
Keyword(s):  
Specific Protein ◽  
Protein Glycosylation ◽  
Site Specific ◽  
Isomer Differentiation

scholarly journals Sensitive and fast characterization of site-specific protein glycosylation with capillary electrophoresis coupled to mass spectrometry

Talanta ◽  
2018 ◽  
Vol 179 ◽  
pp. 22-27 ◽  
Author(s):  
Yanyan Qu ◽  
Liangliang Sun ◽  
Guijie Zhu ◽  
Zhenbin Zhang ◽  
Elizabeth H. Peuchen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Capillary Electrophoresis ◽  
Specific Protein ◽  
Protein Glycosylation ◽  
Site Specific

scholarly journals Multilayered N-glycoproteomics reveals impaired N-glycosylation promoting Alzheimer’s disease

2019 ◽  
Author(s):  
Pan Fang ◽  
Juan-Juan Xie ◽  
Shao-Ming Sang ◽  
Lei Zhang ◽  
Ming-Qi Liu ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Wild Type Mouse ◽  
Specific Protein ◽  
Protein Glycosylation ◽  
Functional Study ◽  
Wild Type ◽  
Site Specific ◽  
Specific Analysis ◽  
Quantitative Analyses

ABSTRACTAlzheimer’s disease (AD) is one of the most common neurodegenerative diseases that currently lacks clear pathogenesis and effective treatment. Protein glycosylation is ubiquitous in brain tissue and site-specific analysis of N-glycoproteome, which is technically challenging, can advance our understanding of the glycoproteins’ role in AD. In this study, we profiled the multilayered variations in proteins, N-glycosites, N-glycans, and in particular site-specific N-glycopeptides in the APP/PS1 and wild type mouse brain through combining pGlyco 2.0 strategy with other quantitative N-glycoproteomic strategies. The comprehensive brain N-glycoproteome landscape was constructed, and rich details of the heterogeneous site-specific protein N-glycosylations were exhibited. Quantitative analyses explored generally downregulated N-glycosylation involving proteins such as glutamate receptors, as well as fucosylated and oligo-mannose type glycans in APP/PS1 mice versus wild type mice. Moreover, our preliminary functional study revealed that N-glycosylation was crucial for the membrane localization of NCAM1 and for maintaining the excitability and viability of neuron cells. Our work offered a panoramic view of the N-glycoproteomes in Alzheimer’s disease and revealed that generally impaired N-glycosylation promotes Alzheimer’s disease progression.

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

2018 ◽  
Author(s):  
Daniel D. Brauer ◽  
Emily C. Hartman ◽  
Daniel L.V. Bader ◽  
Zoe N. Merz ◽  
Danielle Tullman-Ercek ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Modification ◽  
Positive Charge ◽  
Specific Protein ◽  
High Yield ◽  
Site Specific ◽  
Protein Cage ◽  
Protein Carriers ◽  
Site Selective ◽  
Targeted Library

<div> <p>Site-specific protein modification is a widely-used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically-hindered N termini, such as virus-like particles like the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.</p> </div>

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.

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