Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Construction of a Dual Protease Column, Subzero (-30 °C) Chromatography System and Multi-channel Precision Temperature Controller for Hydrogen-Deuterium Exchange Mass Spectrometry.

ABSTRACT: This tutorial provides mechanical drawings, electrical schematics, parts lists, stereolithography (STL) files for producing three-dimensional (3D)-printed parts, initial graphics exchange specification (IGS) files for automated machining, and instructions necessary for construction of a dual protease column, subzero, liquid chromatography system for hydrogen-deuterium exchange mass spectrometry (HDX-MS). Electro-mechanical schematics for construction of two multi-zone temperature controllers that regulate to ±0.05 oC are also included in this tutorial.

SUBMITTER: Hudgens JW

PROVIDER: S-EPMC9097885 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Construction of a Dual Protease Column, Subzero (-30 °C) Chromatography System and Multi-channel Precision Temperature Controller for Hydrogen-Deuterium Exchange Mass Spectrometry.

Hudgens Jeffrey W JW

Journal of research of the National Institute of Standards and Technology 20200812

This tutorial provides mechanical drawings, electrical schematics, parts lists, stereolithography (STL) files for producing three-dimensional (3D)-printed parts, initial graphics exchange specification (IGS) files for automated machining, and instructions necessary for construction of a dual protease column, subzero, liquid chromatography system for hydrogen-deuterium exchange mass spectrometry (HDX-MS). Electro-mechanical schematics for construction of two multi-zone temperature controllers tha ...[more]

PMID: 35573859

Similar Datasets

Hydrophilic Interaction Liquid Chromatography at Subzero Temperature for Hydrogen-Deuterium Exchange Mass Spectrometry.

Project description:Chromatographic separations at subzero temperature significantly improve the precision of back-exchange-corrected hydrogen-deuterium exchange mass spectrometry (HDX-MS) determinations. Our previously reported dual-enzyme HDX-MS analysis instrument used reversed phase liquid chromatography (RPLC) at -30 °C, but high backpressures limited flow rates and required materials and equipment rated for very high pressures. Here, we report the design and performance of a dual-enzyme HDX-MS analysis instrument comprising a RPLC trap column and a hydrophilic interaction liquid chromatography (HILIC) analytical column in a two-dimensional RPLC-HILIC configuration at subzero temperature. During operation at -30 °C, the HILIC column manifests greatly reduced backpressure, which enables faster analytical flow rates and the use of materials rated for lower maximum pressures. The average peptide eluted from a HILIC column during a 40 min gradient at -30 °C contained ≈13% more deuterium than peptides eluted from a tandem RPLC-RPLC apparatus using a conventional 8 min gradient at 0 °C. A subset of peptides eluted from the HILIC apparatus contained ≈24% more deuterium.

| S-EPMC10704588 | biostudies-literature

Subzero temperature chromatography for reduced back-exchange and improved dynamic range in amide hydrogen/deuterium exchange mass spectrometry.

Project description:Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of the deuterium label (back-exchange) during the course of the analysis. A method to limit loss of the label during the separation stage of the analysis using subzero temperature reversed-phase chromatography is presented. The approach is facilitated by the use of buffer modifiers that prevent freezing. We evaluated ethylene glycol, dimethyl formamide, formamide, and methanol for their freezing point suppression capabilities, effects on peptide retention, and their compatibilities with electrospray ionization. Ethylene glycol was used extensively because of its good electrospray ionization compatibility; however, formamide has potential to be a superior modifier if detrimental effects on ionization can be overcome. It is demonstrated using suitable buffer modifiers that separations can be performed at temperatures as low as -30 °C with negligible loss of the deuterium label, even during long chromatographic separations. The reduction in back-exchange is shown to increase the dynamic range of hydrogen/deuterium exchange mass spectrometry in terms of mixture complexity and the magnitude with which changes in deuteration level can be quantified.

| S-EPMC3494095 | biostudies-literature

Chromatography at -30 °C for Reduced Back-Exchange, Reduced Carryover, and Improved Dynamic Range for Hydrogen-Deuterium Exchange Mass Spectrometry.

Project description:For hydrogen-deuterium exchange mass spectrometry (HDX-MS) to have an increased role in quality control of biopharmaceuticals, H for D back-exchange occurring during protein analyses should be minimized to promote greater reproducibility. Standard HDX-MS analysis systems that digest proteins and separate peptides at pH 2.7 and 0 °C can lose >30% of the deuterium marker within 15 min of sample injection. This report describes the architecture and performance of a dual-enzyme, HDX-MS instrument that conducts liquid chromatography (LC) separations at subzero temperature, thereby reducing back-exchange and supporting longer LC separations with improved chromatographic resolution. LC separations of perdeuterated, fully reduced, iodoacetamide-treated BSA protein digest standard peptides were performed at 0, -10, -20, and -30 °C in ethylene glycol (EG)/H2O mixtures. Analyses conducted at -20 and -30 °C produced similar results. After subtracting for deuterium retained in arginine side chains, the average peptide eluted during a 40 min gradient contained ≈16% more deuterium than peptides eluted with a conventional 8 min gradient at 0 °C. A subset of peptides exhibited ≈26% more deuterium. Although chromatographic peaks shift with EG concentration and temperature, the apparatus elutes unbroadened LC peaks. Electrospray ion intensity does not decline with increasing EG fraction. To minimize bias from sample carryover, the fluidic circuits allow flush and backflush cleaning of all enzyme and LC columns. The system can perform LC separations and clean enzyme columns simultaneously. Temperature zones are controlled ±0.058 °C. The potential of increased sensitivity by mixing acetonitrile with the analytical column effluent was also examined.

| S-EPMC9264389 | biostudies-literature

Subzero Celsius separations in three-zone temperature controlled hydrogen deuterium exchange mass spectrometry.

Project description:Hydrogen deuterium exchange mass spectrometry (HDX MS) reports on the conformational landscape of proteins by monitoring the exchange between backbone amide hydrogen atoms and deuterium in the solvent. To maintain the label for analysis, quench conditions of low temperature and pH are required during the chromatography step performed after protease digestion but before mass spectrometry. Separation at 0°C is often chosen as this is the temperature where the most deuterium can be recovered without freezing of the typical water and acetonitrile mobile phases. Several recent reports of separations at subzero Celsius emphasize the promise for retaining more deuterium and using a much longer chromatographic gradient or direct infusion time. Here we present the construction and validation of a modified Waters nanoACQUITY HDX manager with a third temperature-controlled zone for peptide separations at subzero temperatures. A new Peltier-cooled door replaces the door of a traditional main cooling chamber and the separations and trapping column are routed through the door housing. To prevent freezing, 35% methanol is introduced post online digestion. No new pumps are required and online digestion is performed as in the past. Subzero separations, using conventional HPLC column geometry of 3μ m particles in a 1×50mm column, did not result in major changes to chromatographic efficiency when lowering the temperature from 0 to -20°C. There were significant increases in deuterium recovery for both model peptides and biologically relevant protein systems. Given the higher levels of deuterium recovery, expanded gradient programs can be used to allow for higher chromatographic peak capacity and therefore the analysis of larger and more complex proteins and systems.

| S-EPMC5675777 | biostudies-literature

High-throughput hydrogen deuterium exchange mass spectrometry (HDX-MS) coupled with subzero-temperature ultrahigh pressure liquid chromatography (UPLC) separation for complex sample analysis.

Project description:Hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful technique for the characterization of protein dynamics and protein interactions. Recent technological developments in the HDX-MS field, such as sub-zero LC separations, large-scale data analysis tools, and efficient protein digestion methods, have allowed for the application of HDX-MS to the analysis of multi protein systems in addition to pure protein analysis. Still, high-throughput HDX-MS analysis of complex samples is not widespread because the co-elution of peptides combined with increased peak complexity after labeling makes peak de-convolution extremely difficult. Here, for the first time, we evaluated and optimized long gradient subzero-temperature ultra-high-pressure liquid chromatography (UPLC) separation conditions for the HDX-MS analysis of complex protein samples such as E. coli cell lysate digest. Under the optimized conditions, we identified 1419 deuterated peptides from 320 proteins at -10 °C, which is about 3-fold more when compared with a 15-min gradient separation under the same conditions. Interestingly, our results suggested that the peptides eluted late in the gradient are well-protected by peptide-column interactions at -10 °C so that peptides eluted even at the end of the gradient maintain high levels of deuteration. Overall, our study suggests that the optimized, sub-zero, long-gradient UPLC separation is capable of characterizing thousands of peptides in a single HDX-MS analysis with low back-exchange rates. As a result, this technique holds great potential for characterizing complex samples such as cell lysates using HDX-MS.

| S-EPMC8265693 | biostudies-literature

Subzero hydrogen-deuterium exchange CE-MS

Project description:We have performed in solution hydrogen exchange (HDX) on peptide standards and bovine hemoglobin with subsequent quenching, pepsin digestion, and cold capillary electrophoretic separation coupled with mass spectrometry (MS) detection for the benchmarking of a lab built HDX-MS platform. Though neutral coated capillaries that eliminate electroosmotic flow provide fast separations with peak capaci-ties surpassing 200, uncoated capillaries with high electroosmotic flow achieve half as much peak ca-pacity with even faster separations but 30% higher deuterium retention for angiotensin II peptide standard. The data obtained from two different separation conditions on peptic digests of bovine he-moglobin shows strong agreement for relative deuterium uptake between methods and provides data amenable to software such as MS-Studio. Processed data from denatured hemoglobin vs the longest time point in this study (50,000s) also shows agreement with subunit interaction sites determined by crystallographic methods.

2022-12-22 | PXD034245 | Pride

High-Precision, Gas-Phase Hydrogen/Deuterium-Exchange Kinetics by Mass Spectrometry Enabled by Exchange Standards.

Project description:Mass spectrometry (MS) has become a primary tool for identifying and quantifying biological molecules. In combination with other orthogonal techniques, such as gas-phase hydrogen/deuterium exchange (gHDX), MS is also capable of probing the structure of ions. However, gHDX kinetics can depend strongly on many factors, including laboratory temperature, instrumental conditions, and instrument platform selection. These effects can lead to high variability with gHDX measurements, which has hindered the broader adoption of gHDX for structural MS. Here we introduce an approach for standardizing gHDX measurements using cosampled standards. Quantifying the exchange kinetics for analytes relative to the exchange kinetics of the standards results in greater accuracy and precision than the underlying absolute measurements. The standardization was found to be effective for several types of analytes including small molecules and intact proteins. A subset of analytes showed deviations in their standardized exchange profiles that are attributed to field heating and the concomitant conformational isomerization. Inclusion of helium during the gHDX process for collisional cooling helps mitigate such variations in exchange kinetics related to ion heating. We anticipate that the outcomes of this research will enable the broader use of gHDX in MS-based workflows for molecular identification and isomer differentiation.

| S-EPMC7333526 | biostudies-literature

Methods for the Analysis of High Precision Differential Hydrogen Deuterium Exchange Data.

Project description:Hydrogen/deuterium exchange (HDX) mass spectrometry has been widely applied to the characterization of protein dynamics. More recently, differential HDX has been shown to be effective for the characterization of ligand binding. Previously we have described a fully automated HDX system for use as a ligand screening platform. Here we describe and validate the required data analysis workflow to facilitate the use of HDX as a robust approach for ligand screening. Following acquisition of HDX data at a single on-exchange time point (n ? 3), one way analysis of variance in conjunction with the Tukey multiple comparison procedure is used to establish the significance of any measured difference. Analysis results are graphed with respect to a single peptide, ligand or group of ligands, or displayed as an overview within a heat map. For the heat map display, only ?%D values with a Tukey-adjusted P value less than 0.05 are colored. Hierarchical clustering is used to bin compounds with highly similar HDX signatures. The workflow is evaluated with a small data set showing the ligand binding domain (LDB) of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR?) screened against 10 functionally selective ligands. More significantly, data for the vitamin D receptor (VDR) in complex with 87 ligands are presented. To highlight the robustness and precision of our automated HDX platform we analyzed the data from 4191 replicate HDX measurements acquired over an eight month timeframe. Ninety six percent of these measurements were within 10 percent of the mean value. Work has begun to integrate these analysis and graphing components within our HDX software suite.

| S-EPMC3081588 | biostudies-literature

Hydrophilic Interaction Liquid Chromatography-Hydrogen/Deuterium Exchange-Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics.

Project description:Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time-m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (D2O) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as N1-acetylspermidine.

| S-EPMC10932214 | biostudies-literature

Hydrogen/deuterium exchange- and protease digestion-based screening assay for protein-ligand binding detection.

Project description:A protease digestion strategy was incorporated into single-point stability of unpurified proteins from rates of H/D exchange (SUPREX), which is a hydrogen/deuterium (H/D) exchange- and mass spectrometry-based assay for the detection of protein-ligand binding. Single-point SUPREX is an abbreviated form of SUPREX in which protein-ligand binding interactions are detected by measuring the increase in a protein's thermodynamic stability upon ligand binding. The new protease digestion protocol provides a noteworthy increase in the efficiency of single-point SUPREX because peptide masses can be determined with greater precision than intact protein masses in the matrix-assisted laser desorption ionization (MALDI) readout of single-point SUPREX. The protocol was evaluated in test screens on two model protein systems, including cyclophilin A (CypA) and the minor allele variant of human alanine:glyoxylate aminotransferase (AGTmi). The test screening results obtained on both proteins revealed that the peptide readout of the single-point SUPREX-protease digestion protocol was more efficient than the intact protein readout of the original single-point SUPREX protocol at discriminating hits and nonhits. In addition to this improvement in screening efficiency, the protease digestion strategy described here is expected to significantly increase the generality of the single-point SUPREX assay.

| S-EPMC2858457 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data