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PLoS One. 2017 Jul 27;12(7):e0175967. doi: 10.1371/journal.pone.0175967. eCollection 2017.

A modified FASP protocol for high-throughput preparation of protein samples for mass spectrometry.

Author information

1
Genetics & Computational Biology Department, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, 4060, Australia.
2
Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
3
Department of Microbiology, Immunology and Tropical Medicine, School of Medicine and Health Sciences, George Washington University, Washington, DC 20052, United States of America.
4
Research Center for Neglected Diseases of Poverty, School of Medicine and Health Sciences, George Washington University, Washington, DC 20052, United States of America.
5
School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, 4072, Australia.

Abstract

To facilitate high-throughput proteomic analyses we have developed a modified FASP protocol which improves the rate at which protein samples can be processed prior to mass spectrometry. Adapting the original FASP protocol to a 96-well format necessitates extended spin times for buffer exchange due to the low centrifugation speeds tolerated by these devices. However, by using 96-well plates with a more robust polyethersulfone molecular weight cutoff membrane, instead of the cellulose membranes typically used in these devices, we could use isopropanol as a wetting agent, decreasing spin times required for buffer exchange from an hour to 30 minutes. In a typical work flow used in our laboratory this equates to a reduction of 3 hours per plate, providing processing times similar to FASP for the processing of up to 96 samples per plate. To test whether our modified protocol produced similar results to FASP and other FASP-like protocols we compared the performance of our modified protocol to the original FASP and the more recently described eFASP and MStern-blot. We show that all FASP-like methods, including our modified protocol, display similar performance in terms of proteins identified and reproducibility. Our results show that our modified FASP protocol is an efficient method for the high-throughput processing of protein samples for mass spectral analysis.

PMID:
28750034
PMCID:
PMC5531558
DOI:
10.1371/journal.pone.0175967
[Indexed for MEDLINE]
Free PMC Article

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