Probing protein structure by amino acid‐specific covalent labeling and mass spectrometry

Mendoza, Vanessa Leah; Vachet, Richard W.

doi:10.1002/mas.20203

Cited by 327 publications

(463 citation statements)

References 149 publications

(197 reference statements)

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“…The initial success of this experimental strategy, i.e., combing protein modification by DMBNHS with data dependant multistage tandem mass spectrometry, provides confidence for its application in future studies involving the examination of protein structure in larger proteins, as well as for the study of protein-protein and protein-ligand interactions. C (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) A (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62) D (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) F (97-112) E …”

Section: Discussionmentioning

confidence: 99%

“…However, limitations of HDX that can hamper determination of the precise location of exchange sites and therefore limit the spatial resolution that can be obtained, are the back-exchange of deuterium to hydrogen during proteolysis and sample handling before MS analysis [7,8], and hydrogen/deuterium 'scrambling' during CID-MS/MS analysis [9]. Recently, the latter problem has been largely overcome by the use of alternate dissociation techniques such as electron capture dissociation (ECD) [10] and electron-transfer dissociation (ETD) during MS/MS [11].Stable chemical labeling strategies have included (1) nonspecific oxidative modification of protein side-chain functional groups induced by hydroxyl radicals formed via the radiolysis of water by synchrotron X-ray pulses [4], or by photolysis of H 2 O 2 using pulsed UV lasers [5], and (2) specific labeling [6] or cross-linking [12] of Address reprint requests to Dr. …”

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confidence: 99%

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confidence: 99%

“…Stable chemical labeling strategies have included (1) nonspecific oxidative modification of protein side-chain functional groups induced by hydroxyl radicals formed via the radiolysis of water by synchrotron X-ray pulses [4], or by photolysis of H 2 O 2 using pulsed UV lasers [5], and (2) specific labeling [6] or cross-linking [12] of selected amino acid side-chain functional groups within a protein of interest. For both approaches, the reactivity of individual amino acid side chains within the protein of interest is highly dependent on their degree of exposure (i.e., 'accessibility') to the solvent environment.…”

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confidence: 99%

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‘Fixed charge’ chemical derivatization and data dependant multistage tandem mass spectrometry for mapping protein surface residue accessibility

Zhou

Wang

et al. 2010

J. Am. Soc. Mass Spectrom.

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Protein surface accessible residues play an important role in protein folding, protein-protein interactions and protein-ligand binding. However, a common problem associated with the use of selective chemical labeling methods for mapping protein solvent accessible residues is that when a complicated peptide mixture resulting from a large protein or protein complex is analyzed, the modified peptides may be difficult to identify and characterize amongst the largely unmodified peptide population (i.e., the 'needle in a haystack' problem). To address this challenge, we describe here the development of a strategy involving the synthesis and application of a novel 'fixed charge' sulfonium ion containing lysine-specific protein modification reagent, S,S'-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), coupled with capillary HPLC-ESI-MS, automated CID-MS/MS, and data-dependant neutral loss mode MS 3 in an ion trap mass spectrometer, to map the surface accessible lysine residues in a small model protein, cellular retinoic acid binding protein II (CRABP II). After reaction with different reagent:protein ratios and digestion with Glu-C, modified peptides are selectively identified and the number of modifications within each peptide are determined by CID-MS/MS, via the exclusive neutral loss(es) of dimethylsulfide, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility) of the peptide. The observation of these characteristic neutral losses are then used to automatically 'trigger' the acquisition of an MS 3 spectrum to allow the peptide sequence and the site(s) of modification to be characterized. Using this approach, the experimentally determined relative solvent accessibilities of the lysine residues were found to show good agreement with the known solution structure of CRABP II. (J Am Soc Mass Spectrom 2010, 21, 1339 -1351) © 2010 American Society for Mass Spectrometry M ass spectrometry (MS) combined with protein labeling has found increasing utility as an alternative tool to conventional high-resolution methods such as X-ray crystallography or NMR for the examination of higher order protein structure (e.g., tertiary and quaternary), conformation, ligand binding, and dynamics [1]. Although typically providing lower resolution than these more established approaches, MS-based analysis strategies have particular advantages in sensitivity and speed, and the capability of analyzing proteins or protein complexes that are not amenable to crystallization, or that fall outside the mass range routinely accessible by NMR.A variety of MS/protein labeling methods have been developed, and are based on either the use of (1) hydrogen-deuterium exchange (HDX) [2,3] or (2) stable chemical modification [4 -6], before proteolytic digestion and analysis by HPLC-MS and/or tandem mass spectrometry (MS/MS). HDX may potentially be used to probe the entire protein backbone, thereby providing the highest resolution of the MS-based approaches. However, limitations of HDX that can hamper determination...

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Section: Discussionmentioning

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‘Fixed charge’ chemical derivatization and data dependant multistage tandem mass spectrometry for mapping protein surface residue accessibility

Zhou

Wang

et al. 2010

J. Am. Soc. Mass Spectrom.

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“…Covalent labeling represents another important approach for monitoring protein conformations and interactions [14][15][16][17][18][19]. Solvent-accessible side chains can be modified by watersoluble reactive species, whereas buried segments are sterically protected.…”

Section: Introductionmentioning

confidence: 99%

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

Vahidi

Konermann

2016

J. Am. Soc. Mass Spectrom.

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Time (s)0Bleaching of the reporter dye cyanine-5 (Cy5) served as readout of the timedependent radical milieu. Surprisingly, Cy5 oxidation extends over tens of milliseconds. This time range is four orders of magnitude longer than expected from the FPOP literature. We demonstrate that the glutamine scavenger generates metastable secondary radicals in the FPOP solution, and that these radicals lengthen the time frame of Cy5 oxidation. Cy5 and similar dyes are widely used for monitoring the radical dose experienced by proteins in solution. The measured Cy5 kinetics thus strongly suggest that protein oxidation in FPOP extends over a much longer time window than previously thought (i.e., many milliseconds instead of one microsecond). The optical approach developed here should be suitable for assessing the performance of future FPOP-like techniques with improved temporal labeling characteristics.

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Copper(II) partially protects three histidine residues and the N‐terminus of amyloid‐β peptide from diethyl pyrocarbonate (DEPC) modification

2020

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Diethyl pyrocarbonate (DEPC) has been primarily used as a residue‐specific modifying agent to study the role of His residues in peptide/protein and enzyme function; however, its action is not specific, and several other residues can also be modified. In the current study, we monitored the reaction of DEPC with amyloid‐beta (Aβ) peptides and insulin by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and determined the modification sites by electrospray ionization quadrupole time‐of‐flight tandem MS (ESI Q‐TOF MS/MS). Our results indicate that five residues in Aβ1–42 are modified in the presence of 30‐fold molar excess of DEPC. After hydroxylamine treatment (which removes modifications from three His residues), two labels remain bound at the peptide N terminus and Lys16. DEPC treatment of Aβ1–42 promotes peptide aggregation, as monitored through the loss of soluble Aβ42 in a semi‐quantitative MALDI‐TOF MS assay. It has been previously proposed that Cu(II) ions protect Aβ1–16 from DEPC modification through binding to His6. We confirmed that Cu(II) ions decrease the stoichiometry of Aβ1–16 modification with the excess of DEPC being lower as compared to the control, which indicates that Cu(II) protects Aβ from DEPC modification. Sequencing of obtained Cu(II)‐protected Aβ1–16 samples showed that Cu(II) does not protect any residues completely, but partially protects all three His residues and the N terminus. Thus, the protection by Cu(II) ions is not related to specific metal binding to a particular residue (e.g. His6), but rather all His residues and the N terminus are involved in binding of Cu(II) ions. These results allow us to elucidate the effect of DEPC modification on amyloidogenity of human Aβ and to speculate about the role of His residues in these processes.

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Probing protein structure by amino acid‐specific covalent labeling and mass spectrometry

Cited by 327 publications

References 149 publications

‘Fixed charge’ chemical derivatization and data dependant multistage tandem mass spectrometry for mapping protein surface residue accessibility

‘Fixed charge’ chemical derivatization and data dependant multistage tandem mass spectrometry for mapping protein surface residue accessibility

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

Copper(II) partially protects three histidine residues and the N‐terminus of amyloid‐β peptide from diethyl pyrocarbonate (DEPC) modification

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