Proteinlynx global server 3

Peptide maps were database searched in ProteinLynX Global server 3.0 (Waters corporation, Milford, MA) with the following processing and workflow parameters: low and elevated intensity thresholds set to 100.0 and 50.0 counts; intensity threshold sets to 750.0 counts; variable modification: N-terminal methylation; non-specific primary digest reagent; false discovery rate set to 4%. Each fragmentation spectrum was manually inspected for assignment confirmation. The peptide map was refined in DynamX 3.0 (Waters corporation, Milford, MA) with a minimum product per amino-acid value of 0.4. DynamX 3.0 was used to extract the centroid masses; only one unique charge state was considered per peptide and no back-exchange correction was performed. HDX results are reported as relative deuterium exchange level expressed in either mass unit or fractional exchange. Fractional exchange data were calculated by dividing the experimental uptake value by the theoretically maximum number of exchangeable backbone amide hydrogens that could be replaced into each peptide in 78.6% excess deuterium. MEMHDX (Hourdel et al. 2016 (link)) was used to visualize and statistically validate the HDX data (Wald test, p-value < 0.01).

The peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using nanoAcquity UHPLC (Waters) and Q-TOF Premier (Waters) as described earlier [40 (link)] with minor modifications. Samples were separated by BEH130 C18 analytical column (200 mm length, 75 μm diameter, 1.7 μm particle size), using a fast 20 min gradient of 5%–40% acetonitrile with 0.1% formic acid at a flow rate 300 nL/min. The data were recorded in the MSE mode (parallel high and low energy traces without precursor ion selection) and processed using ProteinLynx Global Server 3.0 (Waters). Spectra were searched against wheat proteome sequences downloaded from UniProt in April 2018 (136,892 entries, uniprot.org). Search parameters were as specified in the following chapter for chymotrypsin, but one allowed miscleavage for trypsin and thermolysin. Thermolysin was defined as cutting on N-terminus after alanine, phenylalanine, isoleucine, leucine, methionine, and valine, but not before proline. Identities were accepted if two or more different peptides with a score higher than 95% reliability threshold were matched. Reliability scores were adjusted based on the distribution of target/decoy queries. In the cases when several sequences matched spectra from a single gel spot, we reported accession with the highest number of reliable peptides.

Peptic peptides were identified based on HPLC separation of non-deuterated protein digest using MS^E. MS^E data was analyzed using ProteinLynx global server 3.0 (Waters). Identified peptides were imported with three parallel non-deuterated experiments into Dynamx 3.0 (Waters). Peptides with sufficient signal intensity and confidence were pooled and used for HDX analysis. Deuterium uptake level (#D) for each peptide at each exchange time point was calculated using Dynamx 3.0 to generate uptake curves for each sample. Data obtained was used to make HDX difference charts in which deuterium labeling for each peptide between the reference and oxidized states was compared.

Hydrogen–deuterium exchange was performed using an HDX Manager (Waters, USA) equipped with a LEAP PAL autosampler (LEAP Technologies, USA). 40 µM d-cGrx1 and m-cGrx1 were prepared in 10 mM potassium phosphate pH 7.0, and 5 mM TCEP was added for a reduced-condition sample. The samples were labeled with 15 volumes of deuterated buffer (10 mM potassium phosphate, D₂O pD 7.0) at 20°C and incubated for various time points: 0.33, 10, 60 and 240 min. The exchange was quenched with an equal volume of a prechilled quench buffer (100 mM potassium phosphate, 0.1 M TCEP, 0.4 M guanidine–HCl pH 2.66 at 0°C). The protein was digested on an Enzymate immobilized pepsin column (Waters, USA) and the peptides were trapped on a pre-column (2.1 × 5 mm, ACQUITY BEH VanGuard) and separated using a C18 column (1 × 100 mm, ACQUITY BEH, 1.7 µm; Waters, USA) with a linear gradient of acetonitrile (5–95%) supplemented with 0.1% formic acid. Peptides were analyzed using a SYNAPT G2-Si mass spectrometer with IMS (Waters, USA). The peptic peptides were identified in undeuterated samples with ProteinLynx Global SERVER 3.0 (Waters, USA). To process the HDX-MS data, the amount of deuterium in each peptide was determined by measuring the centroid of the isotopic distribution using DynamX 3.0 (Waters, USA).