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Strategies to enable large-scale proteomics for reproducible research.

ABSTRACT: 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.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Ovary, Prostate Gland

DISEASE(S): Cancer

SUBMITTER: Rebecca Poulos

LAB HEAD: Qing Zhong

PROVIDER: PXD015912 | Pride | 2020-07-20

REPOSITORIES: Pride

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Publications

Strategies to enable large-scale proteomics for reproducible research.

Poulos Rebecca C RC Hains Peter G PG Shah Rohan R Lucas Natasha N Xavier Dylan D Manda Srikanth S SS Anees Asim A Koh Jennifer M S JMS Mahboob Sadia S Wittman Max M Williams Steven G SG Sykes Erin K EK Hecker Michael M Dausmann Michael M Wouters Merridee A MA Ashman Keith K Yang Jean J Wild Peter J PJ deFazio Anna A Balleine Rosemary L RL Tully Brett B Aebersold Ruedi R Speed Terence P TP Liu Yansheng Y Reddel Roger R RR Robinson Phillip J PJ Zhong Qing Q

Nature communications 20200730 1

Reproducible research is the bedrock of experimental science. To enable the deployment of large-scale proteomics, we assess the reproducibility of mass spectrometry (MS) over time and across instruments and develop computational methods for improving quantitative accuracy. We perform 1560 data independent acquisition (DIA)-MS runs of eight samples containing known proportions of ovarian and prostate cancer tissue and yeast, or control HEK293T cells. Replicates are run on six mass spectrometers o ...[more]

PMID: 32732981

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Project description:One µg of total RNA from either an individual rat or from a pooled sample was amplified and labeled with a fluorescent dye (either Cy3 or Cy5) using the Low RNA Input Linear Amplification Labeling kit (Agilent Technologies, Palo Alto, CA) following the manufacturer’s protocol. The amount and quality of the resulting fluorescently labeled cRNA was assessed using a Nanodrop ND-100 spectrophometer and an Agilent Bioanalyzer. Equal amounts of Cy3- or Cy5-labeled cRNA were hybridized to the Agilent Rat Oligo Microarray (Agilent Technologies, Inc., Palo Alto, CA) for 17 hrs, prior to washing and scanning. Data was extracted from the resulting images using Agilent’s Feature Extraction Software (Agilent Technologies, Inc., Palo Alto, CA). For day/night comparisons, two types of complementing hybridizations were performed: hepatic RNA from individual day rats was hybridized against a pool made up of RNA from 12 hour offset night rats, and RNA from individual night rats was hybridized against a pool made up of RNA from 12-hour offset day rats. Specifically, hepatic RNA samples from three individual day rats collected ten hours after light on (CT10) were hybridized against a pooled RNA sample composed of equal aliquots of RNA from the livers of six night rats collected ten hrs after lights off (CT22); and RNA samples from three individual night rats collected at CT22 were hybridized against a pooled RNA sample composed of equal aliquots of RNA from the livers of six day rats collected at CT10. This was replicated in a second study for a total of 24 hybridizations of 12 hour offset samples. For time of day comparisons, equal aliquots of RNA from the livers of six rats collected at each of four different times of the circadian day (CT4, CT10, C16 and CT22) were pooled. The two replicate studies thus resulted in 8 pools (two replicate pools/time point times four time points); each pool was hybridized against a Universal Rat Reference RNA Standard (Stratagene, La Jolla, CA). Keywords: time-course

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Strategies to enable large-scale proteomics for reproducible research.

Publications

Strategies to enable large-scale proteomics for reproducible research.

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