Variable-Velocity Traveling-Wave Ion Mobility Separation Enhancing Peak Capacity for Data-Independent Acquisition Proteomics

Haynes, Sarah E.; Polasky, Daniel A.; Dixit, Sugyan M.; Majmudar, Jaimeen D.; Neeson, Kieran; Ruotolo, Brandon T.; Martin, Brent R.

doi:10.1021/acs.analchem.7b00112

Cited by 81 publications

(88 citation statements)

References 16 publications

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“…Native mass spectra were calibrated externally using a solution of cesium iodide (100 mg/mL) and processed with Masslynx V4.1 software (Waters, Milford, MA). CIU data was extracted using TWIMExtract and analyzed using the CIUSuite to generate CIU plots and perform CIU comparisons. Root‐mean‐square deviation (RMSD) values were computed from a pixel‐to‐pixel comparison of two CIU fingerprints.…”

Section: Methodsmentioning

confidence: 99%

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

et al. 2018

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Antibody–drug conjugates (ADCs) are antibody‐based therapeutics that have proven to be highly effective cancer treatment platforms. They are composed of monoclonal antibodies conjugated with highly potent drugs via chemical linkers. Compared to cysteine‐targeted chemistries, conjugation at native lysine residues can lead to a higher degree of structural heterogeneity, and thus it is important to evaluate the impact of conjugation on antibody conformation. Here, we present a workflow involving native ion mobility (IM)‐MS and gas‐phase unfolding for the structural characterization of lysine‐linked monoclonal antibody (mAb)–biotin conjugates. Following the determination of conjugation states via denaturing Liquid Chromatography‐Mass Spectrometry (LC–MS) measurements, we performed both size exclusion chromatography (SEC) and native IM‐MS measurements in order to compare the structures of biotinylated and unmodified IgG1 molecules. Hydrodynamic radii (Rh) and collision cross‐sectional (CCS) values were insufficient to distinguish the conformational changes in these antibody–biotin conjugates owing to their flexible structures and limited instrument resolution. In contrast, collision induced unfolding (CIU) analyses were able to detect subtle structural and stability differences in the mAb upon biotin conjugation, exhibiting a sensitivity to mAb conjugation that exceeds native MS analysis alone. Destabilization of mAb–biotin conjugates was detected by both CIU and differential scanning calorimetry (DSC) data, suggesting a previously unknown correlation between the two measurement tools. We conclude by discussing the impact of IM‐MS and CIU technologies on the future of ADC development pipelines.

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Section: Methodsmentioning

confidence: 99%

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

et al. 2018

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“…Raw data were analyzed using MassLynx (Waters). ATDs for all CCS calibrants as well as ScSERF complex samples were extracted from raw data using TWIMExtract (49), and IM ATDs were fit to Gaussian functions using CIUSuite2 (50).…”

Section: ½M ½Mmentioning

confidence: 99%

SERF engages in a fuzzy complex that accelerates primary nucleation of amyloid proteins

Meinen

Gadkari

Stull

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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The assembly of small disordered proteins into highly ordered amyloid fibrils in Alzheimer’s and Parkinson’s patients is closely associated with dementia and neurodegeneration. Understanding the process of amyloid formation is thus crucial in the development of effective treatments for these devastating neurodegenerative diseases. Recently, a tiny, highly conserved and disordered protein called SERF was discovered to modify amyloid formation in Caenorhabditis elegans and humans. Here, we use kinetics measurements and native ion mobility-mass spectrometry to show that SERF mainly affects the rate of primary nucleation in amyloid formation for the disease-related proteins Aβ40 and α-synuclein. SERF’s high degree of plasticity enables it to bind various conformations of monomeric Aβ40 and α-synuclein to form structurally diverse, fuzzy complexes. This structural diversity persists into early stages of amyloid formation. Our results suggest that amyloid nucleation is considerably more complex than age-related conversion of Aβ40 and α-synuclein into single amyloid-prone conformations.

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“…Analysis and visualization of nESI(-IM)-MS data was done using Masslynx V4.1 (Waters Corporation, Wilmslow, UK) and Origin 8.0 (OriginLab Corporation, Northampton, MA, USA). Further analysis of CIU plots was performed using TWIMExtract and CIUSuite 2 [ 57 , 58 ].…”

Section: Methodsmentioning

confidence: 99%

Effects of Detergent on α-Synuclein Structure: A Native MS-Ion Mobility Study

Moons

Bruijne

Maudsley

et al. 2020

IJMS

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The intrinsically disordered protein α-synuclein plays a major role in Parkinson’s disease. The protein can oligomerize resulting in the formation of various aggregated species in neuronal cells, leading to neurodegeneration. The interaction of α-synuclein with biological cell membranes plays an important role for specific functions of α-synuclein monomers, e.g., in neurotransmitter release. Using different types of detergents to mimic lipid molecules present in biological membranes, including the presence of Ca2+ ions as an important structural factor, we aimed to gain an understanding of how α-synuclein interacts with membrane models and how this affects the protein conformation and potential oligomerization. We investigated detergent binding stoichiometry, affinity and conformational changes of α-synuclein taking detergent concentration, different detergent structures and charges into account. With native nano-electrospray ionization ion mobility-mass spectrometry, we were able to detect unique conformational patterns resulting from binding of specific detergents to α-synuclein. Our data demonstrate that α-synuclein monomers can interact with detergent molecules irrespective of their charge, that protein-micelle interactions occur and that micelle properties are an important factor.

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Variable-Velocity Traveling-Wave Ion Mobility Separation Enhancing Peak Capacity for Data-Independent Acquisition Proteomics

Cited by 81 publications

References 16 publications

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

SERF engages in a fuzzy complex that accelerates primary nucleation of amyloid proteins

Effects of Detergent on α-Synuclein Structure: A Native MS-Ion Mobility Study

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