Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry

Wang, Guanbo; Jong, Rob N. de; Bremer, Ewald T.J. van den; Parren, Paul W.H.I.; Heck, Albert J. R.

doi:10.1021/acs.analchem.6b05129

Cited by 37 publications

(55 citation statements)

References 41 publications

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“…Although S-RBD glycoforms can be inferred from the timsTOF analysis, we cannot assign the glycan structures or occupancy from only the results presented in Figures 3,4 due to the mass degeneracy and microheterogeneity of O-glycans. 40,62 To achieve in-depth glycoform analysis, we further utilized an ultrahigh-resolution 12T FTICR capable of baseline and isotopically resolving the S-RBD O-glycoforms for MS/MS analysis (see Methods for details, Figure S4). Following further MS 1 analysis, individual glycoforms were directly visualized with high resolution (Figure 5A,B).…”

Section: Comprehensive Characterization Of S-rbd O-glycoforms By Highmentioning

confidence: 99%

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

Roberts

Mann

Melby

et al. 2021

Preprint

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry and the S protein glycosylation is strongly implicated in altering viral binding/function and infectivity. However, the structures and relative abundance of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis. Here, we report the complete structural characterization of intact O-glycan proteoforms using native top-down mass spectrometry (MS). By combining trapped ion mobility spectrometry (TIMS), which can separate the protein conformers of S-RBD and analyze their gas phase structural variants, with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS analysis, the O-glycoforms of the S-RBD are comprehensively characterized, so that seven O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that native top-down MS can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants, as well as other O-glycoproteins in general.

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Section: Comprehensive Characterization Of S-rbd O-glycoforms By Highmentioning

confidence: 99%

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

Roberts

Mann

Melby

et al. 2021

Preprint

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“…Gels I and II were run at room temperature in 4-12% Tris-Glycine Mini polyacrylamide gel (Novex TM TermoFisher Scientific) at 100 V constant and~350 mA using Tris-glycine pH 8.3 as running buffer (Table 1). Coomassie blue gel running time was 2 h. Gels for disaccharidase activity were run for a total of 3 h to have better separation between these enzymes that have similar molecular weight and overlapping activity [8,9,[13][14][15]. The two gels for disaccharidase activity were immersed in either maltose or sucrose 56 mM in maleate-OH buffer for 1 hour at 37 • C ( Table 1).…”

Section: Brush Border Membrane Preparation and Zymographymentioning

confidence: 99%

A Fast and Accurate Method to Identify and Quantify Enzymes in Brush-Border Membranes: In Situ Hydrolysis Followed by Nano LC-MS/MS

Brun

Magallanes

Rio

et al. 2020

MPs

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A simple method for the identification of brush-border membrane α-glucosidases is described. The proteins were first solubilized and separated in a gel under native, non-denaturing, conditions. The gel was then incubated in substrate solutions (maltose or sucrose), and the product (glucose) exposed in situ by the oxidation of o-dianisidine, which yields a brown-orange color. Nano-liquid chromatography coupled to mass spectrometry analyses of proteins (nano LC-MS/MS) present in the colored bands excised from the gels, was used to confirm the presence of the enzymes. The stain is inexpensive and the procedure permits testing several substrates in the same gel. Once enzymes are identified, their abundance, relative to that of other proteins in the brush border, can be semi-quantified using nano LC-MS/MS.

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“…Further, progress in the ability of mass spectrometers to analyze large intact proteins is increasing the utility of this technology for identifying and monitoring functionally relevant product variants. Example include strategies for top down elucidation of site-specific variants as well as additional capabilities for characterizing variants and their impact on protein interactions in intact molecules under native conditions [59][60][61][62][63][64].…”

Section: State-of-the-art Analytics For Detection and Control Of Critmentioning

confidence: 99%

Maintaining consistent quality and clinical performance of biopharmaceuticals

Lamanna¹,

Holzmann

Cohen³

et al. 2018

Expert Opinion on Biological Therapy

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Biopharmaceuticals are large protein based drugs which are heterogeneous by nature due to post translational modifications resulting from cellular production, processing and storage. Changes in the abundance of different variants over time are inherent to biopharmaceuticals due to their sensitivity to subtle process differences and the necessity for regular manufacturing changes. Product variability must thus be carefully controlled to ensure that it does not result in changes in safety or efficacy. Areas covered: The focus of this manuscript is to provide improved understanding of the science and strategies used to maintain the quality and clinical performance of biopharmaceuticals, including biosimilars, throughout their lifecycle. This review summarizes rare historical instances where clinically relevant changes have occurred, defined here as clinical drift, and discusses modern tools used to prevent such changes, including improved analytics, quality systems and regulatory frameworks. Expert opinion: Despite their size complexity and heterogeneity, modern analytics, manufacturing quality systems and comparability requirements for the evaluation of manufacturing changes cumulatively help to ensure the consistent quality and clinical performance of biopharmaceuticals throughout their product lifecycle. Physicians and patients can expect the same safety and efficacy from biopharmaceuticals and their respective biosimilars irrespective of batch or production history.

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Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry

Cited by 37 publications

References 41 publications

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

A Fast and Accurate Method to Identify and Quantify Enzymes in Brush-Border Membranes: In Situ Hydrolysis Followed by Nano LC-MS/MS

Maintaining consistent quality and clinical performance of biopharmaceuticals

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