Probing the Non-covalent Structure of Proteins by Amide Hydrogen Exchange and Mass Spectrometry

Smith, David L.; Deng, Yuzhong; Zhang, Zhongqi

doi:10.1002/(sici)1096-9888(199702)32:2<135::aid-jms486>3.0.co;2-m

Cited by 385 publications

(167 citation statements)

References 18 publications

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“…These electrochemically induced pH changes can cause protein unfolding if time allows [23]. For H/D exchange studies of proteins, the decrease in pH may result in more artifactual exchange [24] if the final pH deviates from the optimum value. Also, possible overlap of the partially deuterated peak with the oxidized peak can lead to imprecise determination of the number of incorporated deuterium labels.…”

Section: Cause Of Oxidation: Origin Of the Oxygen Atommentioning

confidence: 99%

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: Cause Of Oxidation: Origin Of the Oxygen Atommentioning

confidence: 99%

Oxidation Artifacts in the Electrospray Mass Spectrometry of Aβ Peptide

2007

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“…Hydrogen exchange of protons in folded proteins in solution depends on two processes, one being the unfolding event which exposes the amide to solvent, and the other the intrinsic exchange of an exposed amide with bulk solvent [37]. Under most conditions (including physiological conditions), protein refolding rates are much higher than the intrinsic amide exchange rates (a situation commonly known as the EX 2 exchange regime) [2].…”

Section: Protein Dynamics and Ligand Binding-hdx Measurements At Neutmentioning

confidence: 99%

Indirect assessment of small hydrophobic ligand binding to a model protein using a combination of ESI MS and HDX/ESI MS

Kaltashov

Eyles

2003

J. Am. Soc. Mass Spectrom.

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Direct mass spectrometric characterization of interactions between proteins and small hydrophobic ligands often poses a serious problem due to the complex instability in the gas phase. We have developed a method that probes the efficacy of ligand-protein interactions indirectly by monitoring changes in protein flexibility. The latter is assessed quantitatively using a combination of charge state distribution analysis and amide hydrogen exchange under both native and mildly denaturing conditions. The method was used to evaluate binding of a model protein cellular retinoic acid binding protein I to its natural ligand all-trans retinoic acid (RA), isomers 13-cis-and 9-cis-RA, and retinol, yielding the following order of ligand affinities: All-trans RA Ͼ 9-cis RA Ͼ 13-cis RA, with no detectable binding of retinol. This order is in agreement with the results of earlier fluorimetric titration studies. Furthermore, binding energy of the protein to each of retinoic acid isomers was determined based on the measured hydrogen exchange kinetics data acquired under native conditions. (J Am Soc Mass Spectrom 2003, 14, 506 -515)

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“…Effective on-line digestion was accomplished in ϳ12 s. The probe should be of general utility for HDX-MS studies of amyloid fibrils and other protein aggregates. (J Am Soc Mass Spectrom 2007, 18, 208 -217 [1][2][3][4][5][6][7]. The method exploits the fact that amide protons on the backbone of proteins exchange with solvent protons or deuterons at rates that depend on whether they are involved in hydrogen bonds in secondary structural elements such as ␣-helices and ␤-sheets, and/or whether they are sterically shielded from the solvent.…”

mentioning

confidence: 99%

A triaxial probe for on-line proteolysis coupled with hydrogen/deuterium exchange-electrospray mass spectrometry

Chen

Cook

Kheterpal

et al. 2007

J. Am. Soc. Mass Spectrom.

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An on-line proteolysis system utilizing a triaxial electrospray probe was developed to aid localization of the hydrogen-bonding interaction sites in hydrogen/deuterium exchange-mass spectrometry (HDX-MS) studies of A␤ (1-40) fibrils. The probe allows delayed introduction of the organic solvent component needed for stable electrospray, thus enhancing hydrolysis performance relative to that of a coaxial probe. Effective on-line digestion was accomplished in ϳ12 s. The probe should be of general utility for HDX-MS studies of amyloid fibrils and other protein aggregates. (J Am Soc Mass Spectrom 2007, 18, 208 -217 [1][2][3][4][5][6][7]. The method exploits the fact that amide protons on the backbone of proteins exchange with solvent protons or deuterons at rates that depend on whether they are involved in hydrogen bonds in secondary structural elements such as ␣-helices and ␤-sheets, and/or whether they are sterically shielded from the solvent. Analysis normally involves exposing a protein or noncovalent complex to exchange conditions in a D 2 O-based buffer solution, followed by measuring the incorporation of deuterium using methods such as magnetic resonance [1-2], infrared spectrometry [3-4], or mass spectrometry (MS) [5][6][7]. HDX-MS is well-suited to analysis of relatively large and soluble proteins and to protein mixtures [5][6][7].The measured mass shift upon deuteration in HDX-MS analysis provides insight into the overall exchange protection of the entire protein molecule. Determination of specific exposed and protected sites by HDX-MS requires coupling with proteolysis [6,8] and/or tandem mass spectrometry (MS/MS) [9], generally with collision-induced dissociation (CID) or (more recently) electron capture dissociation (ECD) [10]. There is some risk of intramolecular scrambling of deuterium labels during CID [11,12] or ECD [13]. Peptide digestion can offer an alternative or supplement to direct CID or ECD for localizing incorporated deuterium°with°HDX-MS.°Pepsin°has°proven°to°be particularly useful for this purpose because of its good activity at the low pH values used to minimize artifactual hydrogen exchange (spurious incorporation or loss of deuterium) during sample work-up after exchange. A typical protocol involves incubation of deuteriumlabeled proteins with pepsin at about pH 2 for ϳ5 min at 0°C (low-temperature also reduces artifactual exchange). The digest is then subjected to liquid chromatography (LC), with on-line MS or MS/MS analysis to assess the deuterium content of individual digestion products° [3-5,°8].°Relatively°high°quantities°of°pepsin are used to minimize digestion time and the corresponding opportunity for scrambling; this can complicate direct analysis of the hydrolysate without LC separation°(due°to°increased°background)° [14].°Use°of an immobilized enzyme in a continuous flow column can°mitigate°these°problems° [14°-16].We have adapted electrospray HDX-MS methodologies for analysis of the A␤ amyloid fibrils associated with°Alzheimer's°disease° [17][18][19].°To°dissolve°fibrils aft...

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Probing the Non-covalent Structure of Proteins by Amide Hydrogen Exchange and Mass Spectrometry

Cited by 385 publications

References 18 publications

Oxidation Artifacts in the Electrospray Mass Spectrometry of Aβ Peptide

Oxidation Artifacts in the Electrospray Mass Spectrometry of Aβ Peptide

Indirect assessment of small hydrophobic ligand binding to a model protein using a combination of ESI MS and HDX/ESI MS

A triaxial probe for on-line proteolysis coupled with hydrogen/deuterium exchange-electrospray mass spectrometry

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