Restraining expansion of the peak envelope in H/D exchange-MS and its application in detecting perturbations of protein structure/dynamics
Hydrogen/deuterium exchange (H/DX) mass spectrometry (MS) is increasingly applied to problems in protein structural biology in order to map protein dynamics and identify sites of interactions. In theory, an MS-based readout of deuterium label incorporation can overcome the concentration, size, purity, and complexity limitations inherent in NMR-based measurements of exchange; however, in practice, these advantages are reduced due to spectral interference and dilution of the sample in deuterium oxide (D 2O). In this study, we demonstrate that popular H/DX labeling strategies aggravate the interference problem and that significant recovery of spectral capacity may be achieved with a "minimalist" strategy. Simulations of peptide deuteration justify large reductions in the level of D 2O used in labeling experiments, as well as reduced numbers of peaks used in making relative labeling measurements between biochemical states of a protein. To demonstrate the utility of a minimalist approach, calmodulin was interrogated in a bottom-up H/DX-MS workflow, and sensitivity to the addition of Ca (2+) as a structural perturbation was measured as a function of % D 2O and the number of peaks used in quantitating deuteration level. It is shown that high sensitivity to change is preserved with deuteration levels of 5.0 +/- 1.1 (apo-CaM) and 1.4 +/- 1.3% (holo-CaM) using 10% D 2O in the labeling experiment. Further, only two peaks of a peptide peak distribution are needed to sensitively monitor changes in protein structure, dynamics, or both.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2008 |
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| Erschienen: |
2008 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:80 |
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| Enthalten in: |
Analytical chemistry - 80(2008), 18 vom: 15. Sept., Seite 7004-11 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Slysz, Gordon W [VerfasserIn] |
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| Links: |
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| Themen: |
Calcium |
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| Anmerkungen: |
Date Completed 30.10.2008 Date Revised 21.11.2013 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1021/ac800897q |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a Hydrogen/deuterium exchange (H/DX) mass spectrometry (MS) is increasingly applied to problems in protein structural biology in order to map protein dynamics and identify sites of interactions. In theory, an MS-based readout of deuterium label incorporation can overcome the concentration, size, purity, and complexity limitations inherent in NMR-based measurements of exchange; however, in practice, these advantages are reduced due to spectral interference and dilution of the sample in deuterium oxide (D 2O). In this study, we demonstrate that popular H/DX labeling strategies aggravate the interference problem and that significant recovery of spectral capacity may be achieved with a "minimalist" strategy. Simulations of peptide deuteration justify large reductions in the level of D 2O used in labeling experiments, as well as reduced numbers of peaks used in making relative labeling measurements between biochemical states of a protein. To demonstrate the utility of a minimalist approach, calmodulin was interrogated in a bottom-up H/DX-MS workflow, and sensitivity to the addition of Ca (2+) as a structural perturbation was measured as a function of % D 2O and the number of peaks used in quantitating deuteration level. It is shown that high sensitivity to change is preserved with deuteration levels of 5.0 +/- 1.1 (apo-CaM) and 1.4 +/- 1.3% (holo-CaM) using 10% D 2O in the labeling experiment. Further, only two peaks of a peptide peak distribution are needed to sensitively monitor changes in protein structure, dynamics, or both | ||
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| 700 | 1 | |a Schriemer, David C |e verfasserin |4 aut | |
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