Electrospray-assisted modification of proteins: a radical probe of protein structure

Maleknia, Simin D.; Chance, Mark R.; Downard, Kevin M.

doi:10.1002/(sici)1097-0231(19991215)13:23<2352::aid-rcm798>3.0.co;2-x

Cited by 117 publications

(171 citation statements)

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“…Although not investigated in this study, another way of suppressing discharge could be the elimination of electrons produced during discharge by flushing the space around the spray tip with an electron scavenging gas such as SF 6 or CO 2 , or by the addition of a halogenated solvent to the liquid stream [20]. The converse approach of swamping the effect by using O 2 sheath gas (as in "radical probe MS" [6][7][8][9][10]) may complicate interpretation of H/D exchange data.…”

Section: Discussionmentioning

confidence: 99%

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Oxidation Artifacts in the Electrospray Mass Spectrometry of Aβ Peptide

2007

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Gradual corrosion of stainless steel electrospray emitters under conditions of normal use generates surface irregularities that can promote electrical discharge. The increased emission current affects the electrochemical reactions associated with the spray process. When sampling the peptide Aβ(1-40), this is manifest by oxidation of methionine at position 35 to methionine sulfoxide. The resultant mass shift and reduced sensitivity can adversely affect H/D exchange experiments. These effects can be avoided by adding a redox buffer or (preferably) by re-polishing the emitter, especially to a rounded geometry.

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Section: Discussionmentioning

confidence: 99%

“…The converse approach of swamping the effect by using O 2 sheath gas (as in "radical probe MS" [6][7][8][9][10]) may complicate interpretation of H/D exchange data. Electron micrographs of emitter A and mass spectra acquired with it in the triaxial mode after 300 h use: (a) before polishing; (b) after polishing.…”

Section: Discussionmentioning

confidence: 99%

Oxidation Artifacts in the Electrospray Mass Spectrometry of Aβ Peptide

2007

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“…To explore further the interaction between this im-portant protein and the peptide melittin, we have applied an approach developed over a number of years to probe protein structure and interactions using oxygen-based radicals [21][22][23][24][25][26][27][28]. Briefly, proteins or their complexes are treated with a high flux of radicals on millisecond timescales that results in the limited (less than a few percent) oxidation of individual amino acid side chains.…”

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confidence: 99%

“…Briefly, proteins or their complexes are treated with a high flux of radicals on millisecond timescales that results in the limited (less than a few percent) oxidation of individual amino acid side chains. It has been found that where the reaction timescales are short (less than 10 -30 ms), the extent of oxidation at reactive amino acid residue side chains is highly influenced by the accessibility of these sites to the bulk solvent [21][22][23][24][25][26][27][28]. This would not be the case were the structure of the protein or complex to be perturbed.…”

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confidence: 99%

“…Initially, radicals were produced on these fast timescales through the radiolysis of dilute peptide and protein solutions with synchrotron radiation [29]. We have since discovered a more convenient source of radicals through the application of an electrical discharge within the electrospray ion source of a mass spectrometer [21,24,25,27,28]. A similar flux of hydroxyl and other oxygen radicals are produced that react with proteins in solution at the needle tip and within the electrosprayed solution droplets.…”

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confidence: 99%

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Hydroxyl radical probe of the calmodulin-melittin complex interface by electrospray ionization mass spectrometry

Wong

Maleknia

Downard

2005

J. Am. Soc. Mass Spectrom.

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The calcium-dependent interaction of calmodulin and melittin is studied through the application of a radical probe approach in which solutions of the protein and peptide and protein alone are subjected to high fluxes of hydroxyl and other oxygen radicals on millisecond timescales. These radicals are generated by an electrical discharge within an electrospray ion source of a mass spectrometer. Condensation of the electrosprayed droplets followed by proteolytic digestion of both calmodulin and melittin has identified residues in both which participate in the interaction and/or are shielded from solvent within the protein complex. Consistent with other theoretical models and available experimental data, the tryptophan residue of melittin at position 19 is shown to be critical to the formation of the complex with the C-terminal domain of peptide enveloped by and protected from oxidation upon binding to the protein. C almodulin is a ubiquitous eukaryotic protein that regulates the activity of a wide variety of enzymes through its binding to them in the presence of calcium [1,2]. The interaction between calmodulin and a target protein is a key step in many calcium-regulated cellular functions [3,4]. Calmodulin has also been shown to be able to bind a wide range of smaller ligands, including polypeptides, in which the interaction takes place between amino acid side chains [5,6]. Peptides with little sequence homology, however, are capable of binding to calmodulin, though the majority of peptides that do so have a high propensity to form an amphipathic ␣-helix [7,8] and contain a cluster of basic residues.The interaction between calmodulin and the ␣-helical peptide melittin has been the subject of a number of investigations over the years using a range of spectroscopic approaches [9 -17]. Fluorescence and circular dichroism spectroscopy have revealed that in the presence of calcium, both calmodulin and melittin undergo significant structural changes, particularly in the vicinity of tyrosine residues 99 and 138 in calmodulin and at tryptophan 19 in melittin [9]. Small angle X-ray scattering has also identified significant structural changes in calmodulin with its transformation from a dumbbell to globular conformation upon its binding to melittin [13]. Similar conclusions have been drawn from limited proteolysis experiments followed by mass spectrometric analysis [16].Despite an earlier report on the successful growth of crystals of the calmodulin-melittin complex containing bound calcium [18], no X-ray crystallographic data have been made publicly available. X-ray crystal data for other calmodulin-peptide complexes, however, have been obtained [19] and the structure of the calmodulinmelittin complex predicted [16] based on this data and proton NMR studies for the unbound protein [20]. Nonetheless, to-date there exists no high-resolution experimental data for the calmodulin-melittin complex that represents an important model of calmodulin target recognition.To explore further the interaction between this im-

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Contributions of mass spectrometry to structural immunology

Downard

2000

J. Mass Spectrom.

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Mass spectrometry has made important contributions to the field of immunology in the past decade. A variety of mass spectrometric‐based techniques have been applied to study the structures of macromolecules that play a vital role in the immune response. These include traditional molecular mass measurements to identify post‐translational modifications and structural heterogeneity, mass mapping of proteolysis products, sequencing by tandem mass spectrometry and conformational analysis. Antigen–antibody and other immune complexes have been detected by mass spectrometry, providing an avenue to study macromolecular assemblies that are important to immune function. By virtue of the ability of mass spectrometry based techniques to analyze complex biological mixtures, mass spectrometry has also been employed to identify and sequence protein epitopes important in both the humoral and cellular immune responses. This has been achieved through a combination of immunoaffinity and mass spectrometric techniques, and the coupling of high‐performance chromatographs to mass spectrometers. These approaches are important for the identification of pathogens and show promise for the early diagnosis of disease associated with viral and bacterial infection and malignancy. These investigations will enable the mechanisms associated with normal and impaired immune function to be elucidated. Mass spectrometry has been utilized to characterize the structure of peptide mimics, multiple antigenic peptides and other constructs in the design of synthetic immunogens. Information derived from these studies will aid in the development of novel therapeutics and vaccines. Copyright © 2000 John Wiley & Sons, Ltd.

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Electrospray-assisted modification of proteins: a radical probe of protein structure

Cited by 117 publications

References 41 publications

Oxidation Artifacts in the Electrospray Mass Spectrometry of Aβ Peptide

Oxidation Artifacts in the Electrospray Mass Spectrometry of Aβ Peptide

Hydroxyl radical probe of the calmodulin-melittin complex interface by electrospray ionization mass spectrometry

Contributions of mass spectrometry to structural immunology

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