Phosphorylation Driven Motions in the COOH-terminal Src Kinase, Csk, Revealed Through Enhanced Hydrogen–Deuterium Exchange and Mass Spectrometry (DXMS)

Hamuro, Yoshitomo; Wong, Lilly; Shaffer, Jennifer; Kim, Jack S.; Stranz, David D.; Jennings, Patricia A.; Woods, Virgil L.; Adams, Joseph A.

doi:10.1016/s0022-2836(02)01003-3

Cited by 75 publications

(87 citation statements)

References 40 publications

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“…This can be used to increase the resolution of the measured exchange. 22,23 By comparison of the exchange of these peptides it was possible to establish that the amide on Phe244 on the heavy chain did not undergo hydrogen exchange, over the time course of the experiment. This also allows us to establish that the exchange that was observed for peptide HC[242-252] occurred on residues 247 or 249-252.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…In a 4°C cold room, 5 l of each solution was further diluted with 15 l of TBS in a microtiter plate employing multichannel pipettors for simultaneous manipulation. Thirty microliters of a stock exchange quench solution [0.8% formic acid, 1.6 M guanidine hydrochloride (Gdn⅐HCl) was then added to each sample (final concentration 0.5% formic acid, 1.0 M Gdn⅐HCl), samples were transferred to autosampler vials, and were then frozen on dry ice within 1 min after addition of quench solution as described (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). Vials with frozen samples were stored at Ϫ80°C until they were transferred to the dry icecontaining sample basin of the cryogenic autosampler module of a DXMS analysis apparatus designed and operated as described (36)(37)(38).…”

Section: Methodsmentioning

confidence: 99%

“…In brief, samples were melted at 0°C, were proteolyzed for 16 sec by exposure to immobilized pepsin, and fragments were collected on a c18 HPLC column, with subsequent acetonitrile gradient elution. Column effluent was analyzed on both a Thermo Finnigan LCQ electrospray mass spectrometer and a Micromass Q-Tof mass spectrometer, as described (32)(33)(34)(35)(36)(37)(38). The SEQUEST software program (Thermo Finnigan, San Jose, CA) identified the likely sequence of the parent peptide ions and these tentative identifications were confirmed with specialized DXMS data reduction software as described (36)(37)(38).…”

Section: Methodsmentioning

confidence: 99%

“…Building on the pioneering work of Englander and coworkers (14,15,27), we have developed and implemented a number of improvements that have significantly improved throughput, comprehensiveness, and resolution. We term the method employing these enhancements high-throughput and highresolution deuterium exchange MS (DXMS) (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38).…”

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confidence: 99%

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Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS

Pantazatos

Kim

Klock

et al. 2004

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

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142

109

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Crystallographic efforts often fail to produce suitably diffracting protein crystals. Unstructured regions of proteins play an important role in this problem and considerable advantage can be gained in removing them. We have developed a number of enhancements to amide hydrogen͞high-throughput and high-resolution deuterium exchange MS (DXMS) technology that allow rapid identification of unstructured regions in proteins. To demonstrate the utility of this approach for improving crystallization success, DXMS analysis was attempted on 24 Thermotoga maritima proteins with varying crystallization and diffraction characteristics. Data acquisition and analysis for 21 of these proteins was completed in 2 weeks and resulted in the localization and prediction of several unstructured regions within the proteins. When compared with those targets of known structure, the DXMS method correctly localized even small regions of disorder. DXMS analysis was then correlated with the propensity of such targets to crystallize and was further used to define truncations that improved crystallization. Truncations that were defined solely on DXMS analysis demonstrated greatly improved crystallization and have been used for structure determination. This approach represents a rapid and generalized method that can be applied to structural genomics or other targets in a high-throughput manner.

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“…Many enzymes can be crystallized in more than one structural conformation, raising the possibility that the structures with lowered energy barrier may be directly associated with the conversion of substrate to product. Diverse solution methods (5)(6)(7)(8) have also shown that specific regions within the enzyme are in dynamic flux and that the binding of ligands can significantly modify the dynamics in unique ways (9)(10)(11). Although these phenomena are likely to be critical for catalysis, a significant dilemma facing structural biology is to correlate experimentally obtained ''frozen'' structures with a quantitative understanding of the functional mechanism.…”

mentioning

confidence: 99%

Ligand-induced global transitions in the catalytic domain of protein kinase A

Hyeon

Jennings²,

Adams³

et al. 2009

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

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112

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Phosphorylation Driven Motions in the COOH-terminal Src Kinase, Csk, Revealed Through Enhanced Hydrogen–Deuterium Exchange and Mass Spectrometry (DXMS)

Cited by 75 publications

References 40 publications

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

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

Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS

Ligand-induced global transitions in the catalytic domain of protein kinase A

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