Quantitative Evaluation of Native Protein Folds and Assemblies by Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS)

Harris, Mary; Raghavan, Deepika; Borysik, Antoni J.

doi:10.1007/s13361-018-2070-3

Cited by 15 publications

(32 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The surrogates were then submitted for optimization, and the noise-handling capabilities of HDXmodeller were investigated by comparing the outputs of the analogue and smooth digital twins. Surrogate data were prepared from various experimental HDX-MS datasets reporting the relative fractional uptake (RFU) of different peptides recorded at seven different isotope exposure times from 15 s to 8 h. 5 RFU are common units to report HDX-MS data, which scale the experimental mass change of a peptide to the maximum possible mass change, thereby normalizing the signal to a value between 0 and 1. Experimental data were optimized using 100 replicate optimization with the best fitting HDX-MS model taken from these 100 replicates as the data surrogate (Methods section).…”

Section: ■ Resultsmentioning

confidence: 99%

“…Surrogate data were prepared from various experimental HDX-MS datasets reporting the relative fractional uptake (RFU) of different peptides recorded at seven different isotope exposure times from 15 s to 8 h . RFU are common units to report HDX-MS data, which scale the experimental mass change of a peptide to the maximum possible mass change, thereby normalizing the signal to a value between 0 and 1.…”

Section: Resultsmentioning

confidence: 99%

“…Few biophysical techniques are able to capture information on protein conformations as effectively as hydrogen deuterium exchange (HDX). The rate of HDX throughout a protein is characteristic of its unique fold and the dynamic properties of its structural ensemble. − Coupled to mass spectrometry, HDX-MS has particular advantages of throughput, sensitivity, and high protein mass range, but these benefits come at the expense of resolution. , Although every amino acid except proline undergoes HDX, the characterization of residue-resolved exchange kinetics is beyond the capabilities of HDX-MS. And while nonergodic fragmentation can increase the resolution of HDX-MS depending on the ion charge state, the benefits of HDX-MS data modeling can be potentially extended to these acquisition methods also. − Protein HDX behavior is typically limited to the resolution of peptides following acid proteolysis and can only be understood qualitatively by comparing how the peptide mass shifts at any particular isotope exposure time change relative to a control sample such as the wild-type or unliganded protein.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Characterization and Management of Noise in HDX-MS Data Modeling

Salmas

Borysik

2021

Anal. Chem.

View full text Add to dashboard Cite

Quantification of hydrogen deuterium exchange (HDX) kinetics can provide information on the stability of individual amino acids in proteins by finding the degree to which the local backbone environment corresponds to that of a random coil. When characterized by mass spectrometry, extraction of HDX kinetics is not possible because different residue exchange rates become merged depending on the peptides that are formed during proteolytic digestion. We have recently developed an advanced programming tool called HDXmodeller, which enables the exchange rates of individual amino acids to be understood by optimization of low-resolution HDX–mass spectrometry (MS) data. HDXmodeller is also uniquely able to appraise each optimization and quantify the accuracy of modeled exchange rates ab initio using a novel autovalidation method based on a covariance matrix. Here, we address the noise-handling capabilities of HDXmodeller and demonstrate the effectiveness of the algorithm on self-inconsistent datasets. Reference intervals for experimental HDX-MS data are also derived, and this information is presented in an updated online workflow for HDXmodeller, allowing users to evaluate the consistency of their data. The development of a modified version of HDXmodeller is also discussed with enhanced noise-handling capability brought about through loss function optimization. Changes in optimizer accuracy with different loss functions are also demonstrated along with the effectiveness of HDXmodeller to select the most effective optimizer for different data using currently embedded autovalidation criteria.

show abstract

Section: ■ Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Characterization and Management of Noise in HDX-MS Data Modeling

Salmas

Borysik

2021

Anal. Chem.

View full text Add to dashboard Cite

show abstract

“…Reference HDX-MS data were prepared using previously obtained HDX-MS peptides maps of alpha lactalbumin, barnase, enolase, and serum amyloid P component with lnP simulated according to previously described methods 22 . To prepare reference HDX-MS data lnP values were first simulated from protein structures according to an in-house version of a well-known expression of protein HDX behaviour 3 , 23 .…”

Section: Methodsmentioning

confidence: 99%

HDXmodeller: an online webserver for high-resolution HDX-MS with auto-validation

Salmas

Borysik

2021

Commun Biol

View full text Add to dashboard Cite

The extent to which proteins are protected from hydrogen deuterium exchange (HDX) provides valuable insight into their folding, dynamics and interactions. Characterised by mass spectrometry (MS), HDX benefits from negligible mass restrictions and exceptional throughput and sensitivity but at the expense of resolution. Exchange mechanisms which naturally transpire for individual residues cannot be accurately located or understood because amino acids are characterised in differently sized groups depending on the extent of proteolytic digestion. Here we report HDXmodeller, the world’s first online webserver for high-resolution HDX-MS. HDXmodeller accepts low-resolution HDX-MS input data and returns high-resolution exchange rates quantified for each residue. Crucially, HDXmodeller also returns a set of unique statistics that can correctly validate exchange rate models to an accuracy of 99%. Remarkably, these statistics are derived without any prior knowledge of the individual exchange rates and facilitate unparallel user confidence and the capacity to evaluate different data optimisation strategies.

show abstract

“…√ Increased pressure on‐column by increasing flow or using a back‐pressure regulator . So far only possible on BEH pepsin columns.…”

Section: Introduction: Structural Biology Of Membrane Proteinsmentioning

confidence: 99%

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

Martens

Politis

2020

Protein Science

View full text Add to dashboard Cite

Integral membrane proteins (IMPs) control countless fundamental biological processes and constitute the majority of drug targets. For this reason, uncovering their molecular mechanism of action has long been an intense field of research.They are, however, notoriously difficult to work with, mainly due to their localization within the heterogeneous of environment of the biological membrane and the instability once extracted from the lipid bilayer. High-resolution structures have unveiled many mechanistic aspects of IMPs but also revealed that the elucidation of static pictures has limitations. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has recently emerged as a powerful biophysical tool for interrogating the conformational dynamics of proteins and their interactions with ligands. Its versatility has proven particularly useful to reveal mechanistic aspects of challenging classes of proteins such as IMPs. This review recapitulates the accomplishments of HDX-MS as it has matured into an essential tool for membrane protein structural biologists. K E Y W O R D Sallosteric coupling, conformational dynamics, GPCRs, hydrogen-deuterium exchange mass spectrometry, integral membrane proteins, transporters Standard: LC: Reverse-phase chromatography on C18 column with prior trapping for desalting. MS: Often Synapt or Xevo-G2 in MS E mode √ Drift-time aligned MS E (HDMS E ) 17 √ LC column with shorter alkyl chain-for example, BEH C4 or C8 13 √ Gradient optimization (8-30%, 8-50%) 15 √ Saw-tooth gradient after peptides elution to prevent carryover 16 Miscellaneous observationsUse of PEG-based detergents (e.g., Triton X-100) complicates peptides identification. Good practice to start with a benchmark condition-for example, locked protein. 16,18,19 Adding TCEP helps but too much (>1 M) reduces spectral quality. 15Note: The tick indicates parameters that have shown improvement compared with the standard conditions.

show abstract

Quantitative Evaluation of Native Protein Folds and Assemblies by Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS)

Cited by 15 publications

References 23 publications

Characterization and Management of Noise in HDX-MS Data Modeling

Characterization and Management of Noise in HDX-MS Data Modeling

HDXmodeller: an online webserver for high-resolution HDX-MS with auto-validation

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

Contact Info

Product

Resources

About