Assessment of the repeatability and reproducibility of hydrogen/deuterium exchange mass spectrometry measurements

Burkitt, William; O’Connor, Gavin

doi:10.1002/rcm.3794

Cited by 53 publications

(65 citation statements)

References 31 publications

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“…All experiments were performed in duplicate. The error of measuring the mass of each peptide was ± 0.20 Da in this experimental setup, consistent with previously obtained values [37][38][39]. Deuterium uptake was calculated by subtraction of the centroid of the isotopic distribution for peptide ions from undeuterated protein from the centroid of the isotopic distribution for peptide ions from the deuterium-labeled sample.…”

Section: Chromatography and Mssupporting

confidence: 86%

Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

Huang

Iacob

Krystek

et al. 2016

J. Am. Soc. Mass Spectrom.

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Abstract. Aggregation of protein therapeutics has long been a concern across different stages of manufacturing processes in the biopharmaceutical industry. It is often indicative of aberrant protein therapeutic higher-order structure. In this study, the aggregation propensity of a human Fc-fusion protein therapeutic was characterized. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied to examine the conformational dynamics of dimers collected from a bioreactor. HDX-MS data combined with spatial aggregation propensity calculations revealed a potential aggregation interface in the Fc domain. This study provides a general strategy for the characterization of the aggregation propensity of Fc-fusion proteins at the molecular level.

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Section: Chromatography and Mssupporting

confidence: 86%

Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

Huang

Iacob

Krystek

et al. 2016

J. Am. Soc. Mass Spectrom.

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“…The cumulative error of measuring deuterium uptake in these assays is B±0.20 Da. Any differences larger than this value were considered significant for the purposes of comparing the datasets 28 . The location of each peptide, according to the labels, is shown on the crystal structures in (c).…”

Section: Discussionmentioning

confidence: 99%

Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors

et al. 2012

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Selective inhibition of protein methyltransferases is a promising new approach to drug discovery. An attractive strategy towards this goal is the development of compounds that selectively inhibit binding of the cofactor, S-adenosylmethionine, within specific protein methyltransferases. Here we report the three-dimensional structure of the protein methyltransferase DOT1L bound to EPZ004777, the first S-adenosylmethionine-competitive inhibitor of a protein methyltransferase with in vivo efficacy. This structure and those of four new analogues reveal remodelling of the catalytic site. EPZ004777 and a brominated analogue, SGC0946, inhibit DOT1L in vitro and selectively kill mixed lineage leukaemia cells, in which DOT1L is aberrantly localized via interaction with an oncogenic MLL fusion protein. These data provide important new insight into mechanisms of cell-active S-adenosylmethioninecompetitive protein methyltransferase inhibitors, and establish a foundation for the further development of drug-like inhibitors of DOT1L for cancer therapy.

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“…27 Briefly, the system used a HTS-PAL robotic autosampler to perform solvent manipulations, columns and switching valves were maintained at 0 C using a cooled water bath. Samples were exchanged in D 2 O for 30 s, 100 s, 300 s, 1000 s, 3000 s, 10,000 s or 30,000 s (10 lL of sample of concentration 10 mg ml À1 was mixed with 190 lL of D 2 O for each analysis).…”

Section: Automated Hydrogen/deuterium Exchange Analysismentioning

confidence: 99%

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

Burkitt¹,

Domann²,

O’Connor³

2010

Protein Science

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Oxidation of methionine residues in biopharmaceuticals is a common and often unwanted modification that frequently occurs during their manufacture and storage. It often results in a lack of stability and biological function of the product, necessitating continuous testing for the modification throughout the product shelf life. A major class of biopharmaceutical products are monoclonal antibodies (mAbs), however, techniques for their detailed structural analysis have until recently been limited. Hydrogen/deuterium exchange mass spectrometry (HXMS) has recently been successfully applied to the analysis of mAbs. Here we used HXMS to identify and localise the structural changes that occurred in a mAb (IgG1) after accelerated oxidative stress. Structural alterations in a number of segments of the Fc region were observed and these related to oxidation of methionine residues. These included a large change in the hydrogen exchange profile of residues 247-253 of the heavy chain, while smaller changes in hydrogen exchange profile were identified for peptides that contained residues in the interface of the C H 2 and C H 3 domains.

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Assessment of the repeatability and reproducibility of hydrogen/deuterium exchange mass spectrometry measurements

Cited by 53 publications

References 31 publications

Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

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