Conformational Analysis of Large and Highly Disulfide-Stabilized Proteins by Integrating Online Electrochemical Reduction into an Optimized H/D Exchange Mass Spectrometry Workflow

Trabjerg, Esben; Jakobsen, Rasmus U.; Mysling, Simon; Christensen, Søren; Jørgensen, Thomas J. D.; Rand, Kasper D.

doi:10.1021/acs.analchem.5b01996

Cited by 44 publications

(37 citation statements)

References 55 publications

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“…The quenched samples were thawed and injected onto a cooled (0°C) reverse-phase UPLC-HDX system (Waters Inc.) with an integrated electrochemical reduction cell (high-pressure -prep cell, internal volume of 11 l, Antec) (46,70,71) and a home-packed pepsin column (internal volume of 60 l). The deuterated protein samples were subjected to online electrochemical reduction and subsequent pepsin digestion at 20°C (46). The resulting peptic peptides were trapped unto a C18 trap column (ACQUITY UPLC BEH C18 1.7 M Van-Guard column, Waters Inc.) and desalted for 6 min at 50 l/min with 0.23% formic acid.…”

Section: Discussionmentioning

confidence: 99%

“…This resulted in low sequence coverage in major parts of NGF and the mature part of proNGF, e.g. total sequence coverage below 50% (46). To meet this challenge, a HDX-MS-compatible workflow employing online electrochemical reduction was developed, allowing us to successfully reduce the cysteine knot under quenched conditions (e.g.…”

Section: Hdx-ms Analysis Of Prongf and Ngfmentioning

confidence: 99%

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Conformational characterization of nerve growth factor-β reveals that its regulatory pro-part domain stabilizes three loop regions in its mature part

Trabjerg

Kartberg

Christensen

et al. 2017

Journal of Biological Chemistry

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Nerve growth factor-β (NGF) is essential for the correct development of the nervous system. NGF exists in both a mature form and a pro-form (proNGF). The two forms have opposing effects on neurons: NGF induces proliferation, whereas proNGF induces apoptosis via binding to a receptor complex of the common neurotrophin receptor (p75NTR) and sortilin. The overexpression of both proNGF and sortilin has been associated with several neurodegenerative diseases. Insights into the conformational differences between proNGF and NGF are central to a better understanding of the opposing mechanisms of action of NGF and proNGF on neurons. However, whereas the structure of NGF has been determined by X-ray crystallography, the structural details for proNGF remain elusive. Here, using a sensitive MS-based analytical method to measure the hydrogen/deuterium exchange of proteins in solution, we analyzed the conformational properties of proNGF and NGF. We detected the presence of a localized higher-order structure motif in the pro-part of proNGF. Furthermore, by comparing the hydrogen/deuterium exchange in the mature part of NGF and proNGF, we found that the presence of the pro-part in proNGF causes a structural stabilization of three loop regions in the mature part, possibly through a direct molecular interaction. Moreover, using tandem MS analyses, we identified two -linked and two-linked glycosylations in the pro-part of proNGF. These results advance our knowledge of the conformational properties of proNGF and NGF and help provide a rationale for the diverse biological effects of NGF and proNGF at the molecular level.

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Section: Discussionmentioning

confidence: 99%

Section: Hdx-ms Analysis Of Prongf and Ngfmentioning

confidence: 99%

Conformational characterization of nerve growth factor-β reveals that its regulatory pro-part domain stabilizes three loop regions in its mature part

Trabjerg

Kartberg

Christensen

et al. 2017

Journal of Biological Chemistry

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“…The quenching buffer usually also contains a denaturing agent (like guanidine hydrochloride) and a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)) to break the disulfide bonds [60]. Reducing agents often work sub-optimally under the conditions of HDX experiments; online electrochemical reduction of disulfides has proved to be a better solution in various applications [61,62]. (This method is now commercially available).…”

Section: Workflow and Equipmentmentioning

confidence: 99%

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

Ozohanics

Ambrus

2020

Life

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Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information on individual proteins as well as large protein complexes. Owing to recent methodological advancements and improving availability of instrumentation, HDX-MS is becoming a routine technique for some applications. When dealing with samples of low to medium complexity and sizes of less than 150 kDa, conformation and ligand interaction analyses by HDX-MS are already almost routine applications. This is also well supported by the rapid evolution of the computational (software) background that facilitates the analysis of the obtained experimental data. HDX-MS can cope at times with analytes that are difficult to tackle by any other approach. Large complexes like viral capsids as well as disordered proteins can also be analyzed by this method. HDX-MS has recently become an established tool in the drug discovery process and biopharmaceutical development, as it is now also capable of dissecting post-translational modifications and membrane proteins. This mini review provides the reader with an introduction to the technique and a brief overview of the most common applications. Furthermore, the most challenging likely applications, the analyses of glycosylated and membrane proteins, are also highlighted.

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“…The RP LC-MS experiments were performed on Agilent 1290 Infinity II coupled with a Thermo Q Exactive Biopharma ( Figure S6) by following previously established protocols. [71][72][73] A solution of TCEP hydrochloride was prepared in water and 0.1% FA at a concentration of 30 mg/mL. For two runs of 100 µg of mAb, the main peak and additional basic peak were separately collected into two Zorbax 300SB-C8 trap columns (Agilent, 1.0 × 50 mm, 3.5 micron) based on UV (280 nm) CEX chromatogram using identical conditions (column, gradient, and buffers).…”

Section: Rp Lc-ms With Online Reduction and Top-down Analysismentioning

confidence: 99%

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

Shi

Xiao

Dillon

et al. 2020

mAbs

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2020) Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis, mAbs, 12:1, 1739825, ABSTRACT Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatographytop-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping. ARTICLE HISTORY

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Conformational Analysis of Large and Highly Disulfide-Stabilized Proteins by Integrating Online Electrochemical Reduction into an Optimized H/D Exchange Mass Spectrometry Workflow

Cited by 44 publications

References 55 publications

Conformational characterization of nerve growth factor-β reveals that its regulatory pro-part domain stabilizes three loop regions in its mature part

Conformational characterization of nerve growth factor-β reveals that its regulatory pro-part domain stabilizes three loop regions in its mature part

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

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