Samples were thawed for 5 min on ice and manually injected into a Waters HDX Manager kept at 1°C. Whole-virus HA samples were trapped on a Waters ACQUITY UPLC CSH C18 VanGuard, 130 Å, 1.7 μm, 2.1 mm by 5 mm trap column for 3 min with a flow of solvent A (2% acetonitrile, 0.1% FA, and 0.025% trifluoroacetic acid) at a rate of 150 μl/min. BHA samples were digested online with immobilized pepsin for 5 min and trapped as described previously (51 (link)). Peptides were resolved over a Waters ACQUITY UPLC CSH C18, 130 Å, 1.7 μm, 1 mm by 100 mm column using a 10-min linear gradient of 3 to 50% solvent B (solvent B: 100% acetonitrile and 0.1% FA) and analyzed using a Waters Synapt G2-Si Q-TOF with ion mobility enabled. Source and desolvation temperatures were 70° and 130°C, respectively. The StepWave ion guide settings were tuned to prevent nonuniform gas-phase proton exchange in the source (52 (link)). A series of trap column wash steps were implemented between each injection to minimize carryover (53 (link)).
Protocol detail
Virus HA Protein Structural Analysis
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Corresponding Organization : University of Washington
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Variable analysis
independent variables
- Thawing time of samples (5 min)
- Injection of samples into Waters HDX Manager
- Digestion of BHA samples with immobilized pepsin (5 min)
- Peptide resolution over a Waters ACQUITY UPLC CSH C18 column
- Peptide analysis using a Waters Synapt G2-Si Q-TOF with ion mobility enabled
- Temperature of Waters HDX Manager (1°C)
- Trap column (Waters ACQUITY UPLC CSH C18 VanGuard, 130 Å, 1.7 μm, 2.1 mm by 5 mm)
- Flow rate of solvent A (150 μl/min)
- Linear gradient of solvent B (3 to 50% over 10 min)
- Source and desolvation temperatures (70°C and 130°C, respectively)
- StepWave ion guide settings to prevent non-uniform gas-phase proton exchange in the source
- Not explicitly mentioned
- Not explicitly mentioned
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