Hydrogen Exchange Mass Spectrometry for the Analysis of Ligand Binding and Protein Aggregation

Zhang, Ying; Rempel, Don L.; Gross, Michael L.

doi:10.1002/9781118703748.ch11

Cited by 4 publications

(5 citation statements)

References 69 publications

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“…Alterations in conformational dynamics upon ligand binding were described in several earlier reports [11,12,14,17,19,[21][22][23]25,126,130]; in selected studies, an allosteric effect could also be identified. A great example of perturbed conformational dynamics was published by Zhou et al in 2017 [25], where HDX-MS analysis shed light on the role of conformational dynamics, which was considerably altered upon ligand binding, in the mechanism of action of the DXPS enzyme; the closed conformation of DXPS proved to be critical for stabilization of the transition state, whereas the open conformation is apparently required for releasing the lactyl-thiamin diphosphate final product.…”

Section: Analysis Of Protein Interactionsmentioning

confidence: 90%

“…Last year, a group of eminent researchers in the field formulated clear recommendations for performing and interpreting HDX-MS experiments [ 4 ] and hence paved the way towards truly standardized experiments. The applications of HDX-MS are very versatile: they include studies of protein complexes [ 5 , 6 , 7 , 8 , 9 , 10 ], ligand binding [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ], dynamic properties like conformational changes and folding/unfolding/refolding [ 26 ], conformational changes that arise from allosteric effects [ 27 , 28 , 29 ], structure and stability of biopharmaceuticals [ 17 , 30 , 31 ] and epitopes [ 15 , 32 , 33 ], etc. Unlike several other biophysical techniques, HDX-MS possesses the advantages of no or very high size limit [ 8 ] and is useful for studying individual proteins as well as large complexes [ 34 , 35 ].…”

Section: Introductionmentioning

confidence: 99%

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Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

Ozohanics

Ambrus

2020

Life

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Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information on individual proteins as well as large protein complexes. Owing to recent methodological advancements and improving availability of instrumentation, HDX-MS is becoming a routine technique for some applications. When dealing with samples of low to medium complexity and sizes of less than 150 kDa, conformation and ligand interaction analyses by HDX-MS are already almost routine applications. This is also well supported by the rapid evolution of the computational (software) background that facilitates the analysis of the obtained experimental data. HDX-MS can cope at times with analytes that are difficult to tackle by any other approach. Large complexes like viral capsids as well as disordered proteins can also be analyzed by this method. HDX-MS has recently become an established tool in the drug discovery process and biopharmaceutical development, as it is now also capable of dissecting post-translational modifications and membrane proteins. This mini review provides the reader with an introduction to the technique and a brief overview of the most common applications. Furthermore, the most challenging likely applications, the analyses of glycosylated and membrane proteins, are also highlighted.

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Section: Analysis Of Protein Interactionsmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

Ozohanics

Ambrus

2020

Life

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“…The change in deuterium uptake was plotted as a function of the ligand-to-protein ratio to obtain a PLIMSTEX titration curve. The data for four peptides from the EF-hands were then fit to a mathematical model to obtain the binding affinities for Ca 2+ binding to CP-Ser by using a nonlinear least-squares (NLLS) regression analysis, as described previously. , Several different mathematical models were fit to the PLIMSTEX and native MS data to arrive at a best-fit model (see Supporting Discussion of the SI).…”

Section: Experimental Methodsmentioning

confidence: 99%

Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry

et al. 2020

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Although knowledge of the coordination chemistry and metal-withholding function of the innate immune protein human calprotectin (hCP) has broadened in recent years, understanding of its Ca2+-binding properties in solution remains incomplete. In particular, the molecular basis by which Ca2+ binding affects structure and enhances the functional properties of this remarkable transition-metal-sequestering protein has remained enigmatic. To achieve a molecular picture of how Ca2+ binding triggers hCP oligomerization, increases protease stability, and enhances antimicrobial activity, we implemented a new integrated mass spectrometry (MS)-based approach that can be readily generalized to study other protein–metal and protein–ligand interactions. Three MS-based methods (hydrogen/deuterium exchange MS kinetics; protein–ligand interactions in solution by MS, titration, and H/D exchange (PLIMSTEX); and native MS) provided a comprehensive analysis of Ca2+ binding and oligomerization to hCP without modifying the protein in any way. Integration of these methods allowed us to (i) observe the four regions of hCP that serve as Ca2+-binding sites, (ii) determine the binding stoichiometry to be four Ca2+ per CP heterodimer and eight Ca2+ per CP heterotetramer, (iii) establish the protein-to-Ca2+ molar ratio that causes the dimer-to-tetramer transition, and (iv) calculate the binding affinities associated with the four Ca2+-binding sites per heterodimer. These quantitative results support a model in which hCP exists in its heterodimeric form and is at most half-bound to Ca2+ in the cytoplasm of resting cells. With release into the extracellular space, hCP encounters elevated Ca2+ concentrations and binds more Ca2+ ions, forming a heterotetramer that is poised to compete with microbial pathogens for essential metal nutrients.

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“…Self-association interactions using mass spectrometry, self-titration and H/D exchange (SIMSTEX) Subsequent work on PLIMSTEX focused on extending the technique to probe oligomerization of proteins. This approach, SIMSTEX is very similar to PLIMSTEX except that the protein is titrated with itself instead of a ligand (Chitta et al, 2006;Zhang et al, 2016). The first demonstration of SIMSTEX was performed on recombinant (r-) human insulin, a 51 amino acid blood sugar regulating protein that consists of 2 chains (A and B) that are held together by disulfide bonds, and selected analogs and mutants.…”

Section: Plimstex Variantsmentioning

confidence: 99%

“…The rationale behind the inverted approach is that the mass spectrometer can monitor the deuterium exchange of the ligand with higher accuracy and more precision than the large protein. In a typical dPLIMSTEX experiment, the protein‐peptide complex is allowed to equilibrate and the resulting sample is separated into two equal volumes (Tu et al, 2010; Zhang et al, 2016). One aliquot continues to HDX analysis, the other is diluted in aqueous buffer.…”

Section: Interrogating Hos Via Ms‐based Footprintingmentioning

confidence: 99%

The Making of a Footprint in Protein Footprinting: A Review in Honor of Michael L. Gross

McKenzie‐Coe

Shortt

Jones

2020

Mass Spectrometry Reviews

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Within the past decade protein footprinting in conjunction with mass spectrometry has become a powerful and versatile means to unravel the higher order structure of proteins. Footprintingbased approaches has demonstrated the capacity to inform on interaction sites and dynamic regions that participate in conformational changes. These findings when set in a biological perspective inform on protein folding/unfolding, protein-protein interactions, and protein-ligand interactions. In this review, we will look at the contribution of Dr. Michael L. Gross to protein footprinting approaches such as hydrogen deuterium exchange mass spectrometry and hydroxyl radical protein footprinting. This review details the development of novel footprinting methods as well as their applications to study higher order protein structure.

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Hydrogen Exchange Mass Spectrometry for the Analysis of Ligand Binding and Protein Aggregation

Cited by 4 publications

References 69 publications

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry

The Making of a Footprint in Protein Footprinting: A Review in Honor of Michael L. Gross

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