Dataset Information

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

ABSTRACT: 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.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Rattus Norvegicus (rat) Homo Sapiens (human)

TISSUE(S): Testis, Brain, Lung, Hek-293t Cell, Ovarian Serous Carcinoma Cell, Leg Muscle, Kidney

DISEASE(S): Malignant Neoplasm Of Ovary

SUBMITTER: Natasha Lucas

LAB HEAD: Phillip J Robinson

PROVIDER: PXD010688 | Pride | 2019-01-21

REPOSITORIES: Pride

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Publications

A Case Study and Methodology for OpenSWATH Parameter Optimization Using the ProCan90 Data Set and 45 810 Computational Analysis Runs.

Peters Sean S Hains Peter G PG Lucas Natasha N Robinson Phillip J PJ Tully Brett B

Journal of proteome research 20190130 3

In the current study, we show how ProCan90, a curated data set of HEK293 technical replicates, can be used to optimize the configuration options for algorithms in the OpenSWATH pipeline. Furthermore, we use this case study as a proof of concept for horizontal scaling of such a pipeline to allow 45 810 computational analysis runs of OpenSWATH to be completed within four and a half days on a budget of US $10 000. Through the use of Amazon Web Services (AWS), we have successfully processed each of ...[more]

PMID: 30652484

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Project description:Background: Marine biofouling negatively impacts industries reliant upon submerged stationary surfaces. The acorn barnacle Amphibalanus amphitrite is a major biofouler that permanently attaches to a wide array of substrates by producing a proteinaceous adhesive at the surface interface. These interface proteins are difficult to solubilize, and the understanding of what proteins exist in the adhesive has only been expanded with the use of the strong polar solvent hexafluoroisopropanol in combination with standard gel-based sample processing methods for proteomics analysis. Although effective, existing sample processing methods are labor and time intensive, hindering progress in this field. Results: We have developed a more efficient sample processing method by exploiting pressure cycling technology, which aids in protein extraction and digestion for proteomics analysis, and explored the efficacy of multiple solvents in combination with pressure on protein identification. We found that barnacle adhesive proteins can be extracted and digested in the same tube using pressure cycling technology, minimizing sample loss, increasing throughput to 16 concurrently processed samples, and decreasing sample processing time to under 8 hours. Pressure cycling technology methods produced similar proteomes in comparison to previous methods. Two solvents which were ineffective at extracting proteins from the adhesive at ambient pressure (urea and methanol) produced more protein identifications under pressure than hexafluoroisopropanol, leading to the identification and our description of >40 novel proteins at the interface. Many A. amphitrite adhesive proteins have no sequence similarity to publically available proteins, highlighting the unique adherent processes evolved by barnacles. Conclusion: Using pressure cycling technology, we describe methods that produce robust and consistent protein identifications from barnacle adhesive. These methods can be used to examine changes in barnacle adhesive, enabling future research on the effects of environmental and surface substrate composition on barnacle cement, a critical step for both broadening the understanding of barnacle adhesion and the development of new coatings for submerged surfaces to combat hard biofoulants.

Dataset Information

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

Publications

A Case Study and Methodology for OpenSWATH Parameter Optimization Using the ProCan90 Data Set and 45 810 Computational Analysis Runs.

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A Case Study and Methodology for OpenSWATH Parameter Optimization Using the ProCan90 Data Set and 45 810 Computational Analysis Runs.