Data Processing in Bottom‐Up Hydrogen Exchange Mass Spectrometry

Sarpe, Vladimir; Schriemer, David C.

doi:10.1002/9781118703748.ch3

Cited by 5 publications

(6 citation statements)

References 54 publications

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“…HDX-MS data analysis progressively shifts towards automation; however, manual validation is still required [ 81 ]. It is much more challenging to analyze deuteration patterns than to simply report the average number of hydrogens exchanged [ 82 ].…”

Section: Theory Of Hdx-msmentioning

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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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: Theory Of Hdx-msmentioning

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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“…In this case, the HX-MS spectra for peptides in a structural variant spiked reference protein sample will contain separated isotopic spectral profiles (i.e., bimodal distributions) corresponding to the populations of reference protein and structural variant. A single centroid cannot be reliably used to determine the extent of HX for bimodal spectra. , None of the peptide mass spectra for W169G and WT protein samples show any completely separated isotopic spectral profiles (see Figure S6 of the Supporting Information). One of the largest HX differences in our data was 3.9 Da for peptide 351–361 after 30 s of labeling.…”

Section: Resultsmentioning

confidence: 99%

A Structural Variant Approach for Establishing a Detection Limit in Differential Hydrogen Exchange-Mass Spectrometry Measurements

Hageman

Weis

2019

Anal. Chem.

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Hydrogen exchange-mass spectrometry (HX-MS) is widely promoted for its ability to detect subtle perturbations in protein structure, but such perturbations will result in small differences in HX. However, the detection limit of HX-MS has not been widely investigated, nor is there a useful approach for defining the detection limit of HX-MS measurements. In this work, we designed a well-characterized structural variant spiking model to investigate the detection limit of conventional peptide-based HX-MS. The detection limit was challenged by spiking small fractions of a structural variant (modeled using maltose binding protein W169G mutant) into a reference protein (wild-type maltose binding protein). As little as 5% of the structural variant could be detected. The small structural perturbation was not resolvable by far UV circular dichroism, differential scanning calorimetry, or size exclusion chromatography. Furthermore, we validated the ability of the hybrid statistical analysis approach, presented in a companion paper (10.1021/acs.analchem.9b01325), to reliably identify small, significant differences in HX-MS measurements. With our structural variant spiking model, we demonstrate a benchmarking approach for determining a detection limit of HX-MS for detection of changes in higher-order structure that might be encountered in protein structural comparability and similarity assessment applications.

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“…32 Conventional MS software is not well-suited for detection and analysis of the binomial distribution. 33 Since our goal is to use hydrogen exchange to identify peptides that bind to a protein target, we merely need to distinguish differential protection between the bound and unbound states of the peptide. To accomplish this, we have developed a D→H exchange workflow.…”

Section: Resultsmentioning

confidence: 99%

Label-Free, In-Solution Screening of Peptide Libraries for Binding to Protein Targets Using Hydrogen Exchange Mass Spectrometry

Maaty

Weis²

2016

J. Am. Chem. Soc.

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There is considerable interest in the discovery of peptide ligands that bind to protein targets. Discovery of such ligands is usually approached by screening large peptide libraries. However, the individual peptides must be tethered to a tag that preserves their individual identities (e.g. phage display or one-bead one-compound). To overcome this limitation, we have developed a method for screening libraries of label-free peptides for binding to a protein target in solution as a single batch. The screening is based on decreased amide hydrogen exchange by peptides that bind to the target. Hydrogen exchange was measured by mass spectrometry. We demonstrate the approach using a peptide library derived from the E. coli proteome that contained 6664 identifiable features. The library was spiked separately with a peptide spanning the calmodulin binding domain of endothelial nitric oxide synthase (eNOS, 494-513) and a peptide spanning the N-terminal twenty residues of bovine ribonuclease A (S peptide). Human calmodulin and bovine ribonuclease S (RNase S) were screened against the library. Using a novel data analysis workflow we identified the eNOS peptide as the only calmodulin binding peptide and S peptide as the only ribonuclease S binding peptide in the library.

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Data Processing in Bottom‐Up Hydrogen Exchange Mass Spectrometry

Cited by 5 publications

References 54 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

A Structural Variant Approach for Establishing a Detection Limit in Differential Hydrogen Exchange-Mass Spectrometry Measurements

Label-Free, In-Solution Screening of Peptide Libraries for Binding to Protein Targets Using Hydrogen Exchange Mass Spectrometry

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