Joerg Dojahn (Presenter)
Sciex, Germany
Authorship: Joerg Dojahn1; Christie Hunter2; Nick Morrice3
(1) Sciex Germany; (2) Sciex USA; (3) Sciex UK
| Short Abstract Data independent acquisition (DIA) has been used to increase the comprehensiveness of data collection while maintaining high quantitative reproducibility. As this technique increasingly proves to be a solid tool for biomarker research, larger sample sets are being analyzed, driving the need for further improvements for throughput and robustness. Here microflow LC was investigated in combination with SWATH® acquisition on complex matrices, to assess depth of coverage and robustness relative to current nanoflow strategies. This approach provides additional workflow options to researchers with higher throughput and robustness needs, enabling to quantify 4500-5000 proteins with CV <20% on ~150 proteomes per week. |
Long Abstract
Introduction
Data independent acquisition (DIA) strategies have been used to increase the comprehensiveness of data collection while maintaining high quantitative reproducibility. Many labs are now using DIA to perform larger scale quantitative proteomic experiments with solid reproducibility on 1000s of proteins in complex matrices. As this technique increasingly proves to be a solid tool for biomarker research, larger sample sets are being analyzed, driving the need for further investigation of workflow improvements for throughput and robustness. Here microflow LC was investigated in combination with SWATH® acquisition on a number of complex matrices, to assess depth of coverage and robustness relative to current nanoflow strategies.
Methods
Separation of a trypsin digest of HEK cell lysate was performed on a NanoLC™ 425 System (SCIEX) operating in microflow mode using 5 µL/min flow rate (total sample time 60min). Total protein injected on column ranged from 1 – 8 µg. The MS analysis was performed on a TripleTOF® 6600 system (SCIEX), with a TurboV™ Source and 25 ìm I.D. electrospray probe (SCIEX). Variable window SWATH® acquisition methods were built using Analyst® TF Software 1.7. Replicate injections of each acquisition condition were processed using SWATH® 2.0 Software. Results analysis were performed in Excel using the SWATH acquisition Replicates template. All protein and peptide numbers reported were determined at <1%FDR and <20% CV across the 5 replicates collected.
Results
When analyzing larger sample sets, robustness and throughput are key and one way to achieve this is to move up from nanoflow into a higher flow rate regime. In previous quantitative work, we had measured a roughly 3-4x loss in sensitivity when moving from 75µm to 300/500µm ID columns so the 300µm ID column at 5µL/min flow was chosen for this work. A 43 min gradient for a total run time of 1hour per sample was selected as the use case to explore. The acquisition conditions to obtain the best results under these chromatographic conditions were explored. Use of higher sample loads provided progressively more quantified proteins (<1% FDR peptide and <20% CV), up to a load of 8µg (~4x higher than typical nanoflow). When the high resolution MS/MS mode was used (>30000 resolution), a ~5% increase in quantified peptides was observed. Increasing numbers of variable sized Q1 windows (up to 100) also provided increasing numbers of high quality quantified proteins/peptides (~8%). Under the final conditions, ~5000 proteins can be quantified from a cell lysate protein in a 1 hour time frame. In comparison to previous nanoflow results (2-3 µg loads in 3-4 hours), we achieved ~85% of the quantified proteins but with ~400% higher throughput. SWATH® acquisition coupled with microflow chromatography provides an additional workflow option to researchers with higher throughput and robustness needs. When more sample is available to move to the higher flow rate regime, very high reproducibility is achievable with faster run times (up to 150 proteomes per week, while still achieving reasonable depth of coverage.
Conclusion
SWATH® acquisition coupled with microflow chromatography provides additional workflow options to researchers with higher throughput and robustness needs, enabling to analyse ~150 proteomes per week and quantified 4500-5000 proteins with CV <20%.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Sciex |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |