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J Proteome Res. 2018 Nov 28. doi: 10.1021/acs.jproteome.8b00684. [Epub ahead of print]

Accelerated Barocycler Lysis and Extraction Sample Preparation for Clinical Proteomics by Mass Spectrometry.

Author information

1
ProCan, Children's Medical Research Institute, Faculty of Medicine and Health , University of Sydney , Westmead , NSW 2145 , Australia.
2
Centre for Cancer Research , Westmead Institute for Medical Research , Westmead , NSW 2145 , Australia.
3
Cell Signalling Unit , Children's Medical Research Institute, The University of Sydney , Westmead , NSW 2145 , Australia.
4
Faculty of Medicine and Health , The University of Sydney , Westmead , NSW 2145 , Australia.
5
Department of Gynaecological Oncology , Westmead Hospital , Westmead , NSW 2145 , Australia.

Abstract

We have 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 solubilization and reliable, controlled proteolytic digestion. Here, a previously reported PCT based protocol was optimized 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, being ready for MS injection in 3 h compared with 6 h for the conventional urea based method. To validate ABLE, it was applied to a diverse range of rat tissues (kidney, lung, muscle, brain, testis), human HEK 293 cell lines, and human ovarian cancer samples, followed by SWATH-mass spectrometry (SWATH-MS). There were similar numbers of quantified proteins between ABLE-SWATH and the conventional method, with greater than 70% overlap for all sample types, except muscle (58%). The ABLE protocol offers a standardized, high-throughput, efficient, and reproducible proteomic preparation method that when coupled with SWATH-MS has the potential to accelerate proteomics analysis to achieve a clinically relevant turn-around time.

KEYWORDS:

PCT; SWATH; barocycler; cancer; clinical proteomics; mass spectrometry; proteomics; sample preparation; tissue biopsy

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