Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Higher Sensitivity, Data Quality and Throughput for Hydrogen Deuterium Exchange Experiments with Extended Ion Mobility Separation (#359)

Heather Patsiouras 1 , Alexandre F Gomes 2 , Lindsay Morrison 3 , Malcolm Anderson 4 , Colette Quinn 3
  1. Waters Australia, Rydalmere, NSW, Australia
  2. Waters Corporation, Milford, U.S.A
  3. Waters Corporation, Milford, U.S.A
  4. Waters Organisation, Wilmslow

Hydrogen-deuterium exchange has become a standard request for characterization of proteins and protein complexes and in addition to providing dynamic structural insight, its use in high-throughput epitope screening has also gained favor. For complex systems, performance improvements in the LC-MS system, higher sensitivity and/or peak capacity, supports richer and higher quality datasets, which in turn can provide more detailed information to the biological question at hand. The new SELECT SERIESTM Cyclic IMSTM, with enhanced ion mobility and mass resolution, offers improved peak capacity to address these challenges. Here, the performance of the Cyclic IMS system for hydrogen deuterium exchange is demonstrated relative to the SYNAPT G2-SiTM and SYNAPT XSTM, for sample limited applications, and for higher throughput data collection.

 Initial experiments were undertaken to benchmark the performance of the SELECT SERIES Cyclic IMS system relative the Synapt G2-Si and Synapt XS, in terms of improvements in sensitivity and peak capacity. Peptide mapping experiments (no labeling) were used to evaluate the performance comparatively. Improved performance was observed on the Synapt XS and Cyclic IMS relative to the Synapt G2-Si in terms of the number of identified peptides and sequence coverage. Approximately 240, 360, and 560 peptides were identified using the Synapt G2-Si, Synapt XS, and Cyclic IMS, respectively. Novel hardware features of the Cyclic IMS system, such as the XS transfer device and dual gain ADC detector, likely contributed to its improved performance versus Synapt XS. Average sequence coverages for the three instruments were 89.0, 98.5, and 100%.

Next, two types of experiments were performed comparatively on the Cyclic IMS: lowering the protein load to simulate sample-limited conditions; and shortening the gradient time to simulate higher throughput conditions, while mitigating the effects of coelution by using single and double pass cyclic ion mobility. For the reduced sample load, peptide identifications and sequence coverage for phosphorylase B were examined using 8, 16, and 32 µM stock solutions. Better than 90% filtered sequence coverage was obtained for the 16 and 8 µM solutions, 93% and 95%, respectively, indicating that good performance HDX peptide mapping can be achieved with lower concentration solutions.

Finally, a truncated gradient with a 3-minute effective ramp and faster flow rate (60 µL/min) was tested. To fully exploit the tunable mobility resolution of the Cyclic IMS, 1 and 2-pass methods were used for both the 7-minute and 3-minute gradients. Reducing the length of the gradient resulted in a decrease in the number of peptides identified; however excellent sequence coverage of greater than 98% was obtained with the shortened gradient.