H‐D exchange experiments were conducted with a Waters Synapt G2S system. Total volume of 5 μl samples containing 10 μM protein in gel filtration buffer were mixed with 55 μl of the same buffer made with D2O for several deuteration times (0 s, 1 min, 2 min, 5 min, 10 min) at 15°C. The exchange was quenched for 2 min at 1°C with an equal volume of quench buffer (3 M guanidine HCl, 0.1% formic acid). Proteins were cleaved with pepsin and separated by reverse‐phase chromatography, then directed into a Waters SYNAPT G2s quadrupole time‐of‐flight mass spectrometer. Peptides were identified using PLGS version 2.5 (Waters, Inc.), deuterium uptake was calculated using DynamX version 2.0 (Waters Corp.), and uptake was corrected for back‐exchange using DECA.53 Uptake plots were generated in Prism version 8.
Dynamx
Manufactured by Waters Corporation
The DynamX is a versatile laboratory equipment designed for high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) applications. It offers precise separation, detection, and data analysis capabilities for a wide range of analytical tasks.
Automatically generated - may contain errors
Lab products found in correlation
Plgs version 2, by Waters Corporation (2 mentions)
Synapt g2 s system, by Waters Corporation (2 mentions)
Proteinlynx global server, by Waters Corporation (2 mentions)
Synapt g2 s quadrupole time of flight mass spectrometer, by Waters Corporation (1 mentions)
Proteinlynx global server plgs, by Waters Corporation (1 mentions)
Proteinlynx global server 2, by Waters Corporation (1 mentions)
Synapt g2s quadrupole time of flight q tof mass spectrometer, by Waters Corporation (1 mentions)
Hdx manager, by Waters Corporation (1 mentions)
9 protocols using dynamx
1
Protein Deuterium Exchange Mass Spectrometry
2
HDX-MS Analysis of Fts Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peptide peak assignment was performed by the ProteinLynx Global Server (PLGS, version 3.0.2, Waters) with the primary sequence of the target proteins. Only the peptide that matched the following criteria were processed: PLGS score >6, precursor ion (MH+) mass error <10 ppm, and the number of product ions per amino acid >0.33. The HDX-MS data of the free and complexed Fts-proteins were processed by the DynamX (Version 3.0.0, Waters) software, and the results were manually inspected. An E coli proteome (GCF_012978145.1) database was used to validate the peptide assignment. The reference mass of each peptide was generated by the same HDX-MS procedure while replacing all the D2O with deionized water. There is no bimodal distribution in the analyzed peptides. The average deviation of the deuterium uptake was below 0.1 Da. Back exchange level was estimated to be an average rate of 50% by analyzing fully deuterated peptides and was not corrected when carrying out comparisons of deuterium uptake.
3
Δ16-TBEVC Peptide Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To identify Δ16-TBEVC peptides obtained from nondeuterated samples and assign their mass in labeled samples, the ProteinLynx Global SERVER (PLGS; Waters; version 3.0.2)/DynamX (Waters; version 3.0) workflow was performed. PLGS processing parameters for MS/MS data were used as follows: chromatographic peak width—automatic; MS TOF resolution—automatic; lock mass for charge 1 to 556.2771 Da/e; lock mass window—0.25 Da; low energy threshold—135.0 counts; elevated energy threshold–30.0 counts; intensity threshold—750.0 counts, and PLGS workflow parameters were used as follows: searching against fasta file containing normal and reverse sequences of Δ16-TBEVC, Nepenthesine-II (UniProt code: Q766C2), and standard contaminants; peptide tolerance and fragment tolerance—automatic; minimum fragment ion matches per peptide—three; minimum fragment ion matches per protein—seven; minimum peptide matches per protein—one; primary digest reagent—nonspecific; number of missed cleavages—one; fixed modifier reagent—12C d0 C; oxidation of methionines as a variable modifier reagent; FDR of four; monoisotopic mass of peptides with charge 1+; and instrument type ESI-QUAD-TOF.
4
Peptic Peptide Identification and HDX-MS
5
Mass Spectrometry-Based Deuterium Uptake Analysis
6
Hydrogen-Deuterium Exchange Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
H-D exchange experiments were conducted with a Waters Synapt G2S system. 5 μL samples containing 10 μM protein in gel filtration buffer were mixed with 55 μL of the same buffer made with D2O for several deuteration times (0 sec, 1 min, 2 min, 5 min, 10 min) at 15°C. The exchange was quenched for 2 min at 1°C with an equal volume of quench buffer (3M guanidine HCl, 0.1% formic acid). Proteins were cleaved with pepsin and separated by reverse-phase chromatography, then directed into a Waters SYNAPT G2s quadrupole time-of-flight (qTOF) mass spectrometer. Peptides were identified using PLGS version 2.5 (Waters, Inc.), deuterium uptake was calculated using DynamX version 2.0 (Waters Corp.), and uptake was corrected for back-exchange using DECA51 (link). Uptake plots were generated in Prism version 8.
7
HDX-MS Analysis of Chl1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HDX-MS was performed using a Waters HDX manager. Samples were prepared by 10-fold dilutions from 5 μM Chl1 protein in deuterated or nondeuterated buffer containing 150 mM NaCl, 50 mM HEPES pH 7.5, 0.5 mM TCEP. The pH of the sample was reduced to 2.3 and an in-line pepsin-immobilized column at 20°C was used for protein digestion. For labelling experiments, apo protein was incubated for 10 s, 100 s, and 1000 s at room temperature. All HDX-MS experiments were performed in triplicate. Sequence coverage and deuterium uptake were analysed by using ProteinLynx Global Server (Waters) and DynamX (Waters) programs, respectively. For mass correction, Leucine enkephalin at a continuous flow rate of 5 μl min−1 was sprayed. All sample preparation and sample loading performed manually.
8
Targeted ETD Analysis of HDX-Protected Peptides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peptides that showed protection upon compound binding from HDX-MS experiments were subjected to ETD applying a targeted approach. Using an exact mass retention time window, peptides with a charge state of +3 and above were selected for fragmentation by ETD. ETD fragment data were analysed initially using DynamX (Waters), followed by visualisation using Prism 6 (Graphpad) and Pymol.
9
Targeted ETD Analysis of HDX-Protected Peptides
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!