ABLE-SWATH - A Case Study and Methodology for OpenSWATH Parameter Optimization Using the ProCan90 Data Set and 45,810 Computational Analysis Runs

Proteomic methods typically involve lengthy, multi-step sample preparation protocols (14-16 hrs), especially for the lysis and digestion of solid tissue samples or cells. We developed a streamlined proteomic sample preparation protocol termed Accelerated Barocycler Lysis and Extraction (ABLE), that substantially reduces the time and cost of tissue sample processing. ABLE is based on pressure cycling technology (PCT) for rapid tissue solubilisation and reliable, controlled proteolytic digestion. Here, the previously reported PCT protocol was optimised using 1-4 mg biopsy punches from rat kidney. The tissue denaturant urea was substituted with a combination of sodium deoxycholate (SDC) and N-propanol. ABLE produced comparable numbers of protein identifications in half the sample preparation time and with reduced cost, being ready for MS injection in 3 hrs (vs the conventional urea PCT method of 6 hrs). To validate the method, it was applied across a diverse range of rat tissues (kidney, lung, muscle, brain, testis), human HEK 293 cells and ovarian tumours by coupling PCT with SWATH-mass spectrometry (SWATH-MS). There were similar numbers of quantified proteins between ABLE-SWATH and PCT-SWATH methods, with greater than 70% overlap for all sample types, except muscle with 58% overlap. The ABLE tissue processing protocol offers a standardised, high-throughput, efficient and reproducible proteomic preparation method, accelerating sample throughput for any type of MS analysis. Coupled with SWATH-MS, ABLE has the potential to accelerate proteomics analysis towards a clinically relevant turn-around-time.