The combination of microflow LC with SWATH® Acquisition for large scale quantitative proteomics studies is becoming increasingly more widespread, due to the improved robustness and throughput obtained relative to the traditional nanoflow LC approach. High quality quantitative datasets have been generated using a standard 1 hour gradient, demonstrating large numbers of proteins quantified routinely.1
Here, an exploration into the impact of gradient length on proteins identified quantified using data dependent (DDA) and data independent acquisition (DIA) was undertaken to provide researchers expanded workflow options with microflow SWATH acquisition. Using microflow liquid chromatography gradients as short as five minutes (total run time <15mins), acquisition parameters were first optimized then used on multiple TripleTOF® 6600 systems to study impact of shortened separations on number of proteins identified and quantified.
Next the fast methods were applied to a small set of cell lines to further characterize the impact of fast gradients on quantitative accuracy.
MATERIALS AND METHODS
Sample Preparation: Cell lysates (HEK, K562 and yeast) were digested with trypsin using standard protocols. Sample loading of 1 µg of total protein were used for each injection.
Chromatography: A NanoLC™ 425 System plumbed for microflow chromatography was used (5 µµL/min) and operated in trap/elute mode. Column temperature was controlled at 30°C. Gradients of 5, 10, 20, or 45 minutes were tested. More information on LC configuration can be found in the SWATH Performance Kit SOP.2
Mass Spectrometry: All data was acquired using a TripleTOF 6600 System with the Turbo V™ Source equipped with the 25 µm hybrid electrodes for microflow LC or the OptiFlow® TurboV source. SWATH acquisition data were collected using a variety of acquisition strategies. The TOF MS scan was 150 msec and the # of Q1 windows/cycle and accumulation time was varied.
Data Processing: Data independent acquisition (DIA) data was processed using SWATH Acquisition microapp in PeakView® Software 2.2 and the Pan Human Library.3 . Results were evaluated using the SWATH Acquisition Replicates Template.
REFERENCES