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

Potriquet, Jeremy, Laohaviroj, Marut, Bethony, Jeffrey M., and Mulvenna, Jason (2017) A modified FASP protocol for high-throughput preparation of protein samples for mass spectrometry. PLoS ONE, 12 (7). e0175967.

[img]
Preview
PDF (Published Version) - Published Version
Available under License Creative Commons Attribution.

Download (986kB) | Preview
View at Publisher Website: http://doi.org/10.1371/journal.pone.0175...
 
47


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.

Item ID: 54243
Item Type: Article (Research - C1)
ISSN: 1932-6203
Additional Information:

© 2017 Potriquet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funders: National Cancer Institute (NCI), National Institute of Allergy and Infectious Disease (NIAID), National Health and Medical Research Council of Australia (NHMRC)
Projects and Grants: NCI R01CA155297, NIAID Award P50AI098639, NHMRC grant 1051627
Date Deposited: 21 Jun 2018 00:59
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1101 Medical Biochemistry and Metabolomics > 110106 Medical Biochemistry: Proteins and Peptides (incl Medical Proteomics) @ 100%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 100%
Downloads: Total: 47
Last 12 Months: 19
More Statistics

Actions (Repository Staff Only)

Item Control Page Item Control Page