Strategies to enable large-scale proteomics for reproducible research.

An experiment was designed to assess the reproducibility of SWATH-MS measurements collected from different mass spectrometers in a single facility over a period spanning approximately four months. Data were acquired with 90-minute gradient lengths at the Australian Cancer Research Foundation International Centre for the Proteome of Human Cancer (ProCan) on six SCIEX TripleTOF 6600 QTOF mass spectrometers. Multiple replicate aliquots were prepared for eight samples. These comprised a dilution series of ovarian cancer tissue (0%, 3.125%, 6.25%, 12.5%, 25% and 50%) offset by yeast and a fixed proportion (50%) of prostate cancer tissue (Samples 1-6), a 1:1 mix of ovarian cancer tissue and yeast cells (Sample 7), and a human cell line (HEK293T; Sample 8). On each mass spectrometer, sets of 20 replicate aliquots (three aliquots of Samples 2-5, and two aliquots of Samples 1, 6-8) were run during each of thirteen 48-hour periods. Experimental data were acquired in 48-hour time periods on each instrument continuously for eight days (with sets of 20 replicates commencing on days 1, 3, 5 and 7), once per week for the remainder of the month (commencing on days 14, 21 and 28), and then once per month for the remainder of the first three months (commencing on days 56 and 84). After each instrument underwent a major clean, the sets of 20 replicates were again run continuously for a further eight days (commencing on days 101, 103, 105 and 107). Data were therefore acquired during a total of thirteen 48-hour periods over approximately four months, during which time the mass spectrometry facility was fully operational. Mass spectrometer maintenance schedules varied according to each individual instrument's performance, and each instrument commenced data acquisition asynchronously within 28 days from the experiment start.