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

Jeremy Potriquet,Marut Laohaviroj,Jason Mulvenna,Jeffrey M Bethony,Jon M Jacobs

doi:10.1371/journal.pone.0175967

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.

Highlights

Filter-aided sample preparation (FASP) is a method for efficiently generating tryptic peptides from complex protein mixtures prior to mass spectral analysis
Using FASP in 96-well plates equipped with cellulose molecular weight cut-off (MWCO) membranes typically requires centrifugation spin times of an hour or more at 3200 × g for efficient buffer exchange in wash steps [7, 8, 12]
The ability of isopropanol to improve the rate of buffer exchange is likely due to a reduction in surface tension between the aqueous layer and the membrane [13] and alcohol can reduce the critical micelle concentrations (CMC) of detergents [14], affecting both the elimination of the detergent during buffer exchanges and its ability to solubilize proteins, this is likely balanced by the positive effects of urea on the CMC [15]

Summary

Introduction

Filter-aided sample preparation (FASP) is a method for efficiently generating tryptic peptides from complex protein mixtures prior to mass spectral analysis. Modified FASP for high-throughput sample preparation from the National Health and Medical Research Council, Australia (NHMRC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Methods

Results

Conclusion