Deglycosylation induces extensive dynamics changes in α‐amylase revealed by hydrogen/deuterium exchange mass spectrometry

Ji, Chengjie; Wei, Gary

doi:10.1002/rcm.6732

Cited by 5 publications

(4 citation statements)

References 28 publications

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“…In some systems, HDX-MS showed mixed effects with some areas becoming stabilized and other regions showing increased accessibility upon glycan removal. 548 − 551 In other glycoproteins, the only observed changes were increased dynamics through the protein upon glycan removal. 541 , 552 Some studies observed only very minor changes in HDX kinetics despite known effects on overall thermal stability upon glycan removal.…”

Section: Current Uses Of Hdx-msmentioning

confidence: 99%

“…Even with limited sequence coverage, HDX-MS has been a reliable tool for mapping binding interfaces and tracking structural changes upon protein–protein and protein–ligand interactions involving serum glycoproteins ,,− and receptors. ,,, Several studies have also used HDX-MS to assess the influence of glycosylation itself on the overall structure of serum glycoproteins with some interesting variability that reveals a complex relationship between a protein’s glycosylation state and its conformational dynamics. In some systems, HDX-MS showed mixed effects with some areas becoming stabilized and other regions showing increased accessibility upon glycan removal. − In other glycoproteins, the only observed changes were increased dynamics through the protein upon glycan removal. , Some studies observed only very minor changes in HDX kinetics despite known effects on overall thermal stability upon glycan removal . The available studies have thus far revealed a complex and very context-dependent relationship between glycosylation and structural dynamics.…”

Section: Current Uses Of Hdx-msmentioning

confidence: 99%

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Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

et al. 2021

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Solution-phase hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) is a widespread tool for structural analysis across academia and the biopharmaceutical industry. By monitoring the exchangeability of backbone amide protons, HDX-MS can reveal information about higher-order structure and dynamics throughout a protein, can track protein folding pathways, map interaction sites, and assess conformational states of protein samples. The combination of the versatility of the hydrogen/deuterium exchange reaction with the sensitivity of mass spectrometry has enabled the study of extremely challenging protein systems, some of which cannot be suitably studied using other techniques. Improvements over the past three decades have continually increased throughput, robustness, and expanded the limits of what is feasible for HDX-MS investigations. To provide an overview for researchers seeking to utilize and derive the most from HDX-MS for protein structural analysis, we summarize the fundamental principles, basic methodology, strengths and weaknesses, and the established applications of HDX-MS while highlighting new developments and applications.

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Section: Current Uses Of Hdx-msmentioning

confidence: 99%

Section: Current Uses Of Hdx-msmentioning

confidence: 99%

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

et al. 2021

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“…The effects of Endo H trimming of high mannose glycans from α-amylase were studied. 161 Several forms of recombinant glucocerebrosidase, sold as Cerezyme, a treatment for Gaucher’s disease, were investigated by HDX MS, 162 including mutants and ligand-bound forms. The effects of pegylation on the conformation and dynamics of granulocyte colony stimulating factor (G-CSF) were studied.…”

Section: Biopharmaceuticalsmentioning

confidence: 99%

“…The conformation and dynamics of insulin were characterized by HDX MS, , including analyses of various forms/versions of insulin with different therapeutic properties. The effects of Endo H trimming of high mannose glycans from α-amylase were studied . Several forms of recombinant glucocerebrosidase, sold as Cerezyme, a treatment for Gaucher’s disease, were investigated by HDX MS, including mutants and ligand-bound forms.…”

Section: Biopharmaceuticalsmentioning

confidence: 99%

Applications of Hydrogen/Deuterium Exchange MS from 2012 to 2014

2014

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Surface modification of chitosan films with a fibronectin fragment–DNA aptamer complex to enhance osteoblastic cell activity: A mass spectrometry approach probing evidence on protein behavior

Saccani

Parisi

Bergonzi

et al. 2019

Rapid Comm Mass Spectrometry

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Rationale Protein dynamics are fundamental for biological activity. Direct surface functionalization with these biomolecules often creates regions presenting non‐homogeneous and altered protein functionality. Aptamers have been shown to be an effective method to enhance the biocompatibility of biomaterials by acting as docking points for biomolecules capable of improving cell responses and the colonization/proliferation processes. Here mass spectrometry (MS) was successfully applied as an analytical approach to study the interactions between a fibronectin fragment (FN) and anti‐fibronectin aptamers in solution and on a chitosan film. Methods A 30 kDa N‐terminal fibronectin fragment, a ssDNA anti‐fibronectin 40 nucleotide long aptamer and chitosan 95/50 (degree of deacetylation 92.6–97.5%; molecular weight 80–220 kDa) were used. First, the dynamics of the FN in aqueous solution, in the presence or absence of anti‐FN‐specific DNA aptamers, were described. Thus, the same study was carried out on 2% (w/w) chitosan‐based films, functionalized with FN alone or through aptamers as selective spacers. Results Amide hydrogen/deuterium exchange mass spectrometry (HDX‐MS) analysis identified the SYRIGDTWSKKDNRGNL and YRVGDTYERPKDSMI, YVVGETWEKPYQGWMM and WERTYLGNAL fragments as presumably involved in the interaction of FN with aptamers. MS findings indicated the improved functionality of FN on chitosan when aptamers were used as selective spacer, and this may explain why cells preferentially attached to aptamer‐bound FN rather than on chitosan‐FN films. Conclusions Aptamers did not affect the amount of adsorbed FN, but influenced its conformation enhancing its biological activity toward adhesion and growth of osteoblasts. HDX‐MS data allowed the identification of the FN/aptamer interaction regions. Differences in FN flexibility on the chitosan film in the presence or absence of the aptamer were established providing insights at the molecular level to better understand the protein biological functionality on cell proliferation.

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Deglycosylation induces extensive dynamics changes in α‐amylase revealed by hydrogen/deuterium exchange mass spectrometry

Abstract: The overall exchange rate is reduced in the glycosylated state, which indicates rigidity enhancement due to the attached carbohydrates.

Cited by 5 publications

References 28 publications

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

Applications of Hydrogen/Deuterium Exchange MS from 2012 to 2014

Surface modification of chitosan films with a fibronectin fragment–DNA aptamer complex to enhance osteoblastic cell activity: A mass spectrometry approach probing evidence on protein behavior

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