Amicon-adapted enhanced FASP: an in-solution digestion-based alternative sample preparation method to FASP

Abstract

Sample preparation is a crucial step for liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics. Sodium dodecyl sulfate (SDS) is a powerful denaturing detergent that allows for long-term preservation of protein integrity. However, as it inhibits trypsin and interferes with LC-MS/MS analyses, it must be removed from samples prior to these experiments. The Filter-Aided Sample Preparation (FASP) method is actually one of the preferred and simplest methods for such purpose. Nonetheless, there exist great disparities in the quality of outcomes when comparing FASP to other protocols depending on the authors, and recent reports have pointed to concerns regarding its depth of proteome coverage. To address these issues, we propose an Amicon-adapted in-solution-based enhanced FASP (eFASP) approach that relies on current best practices in comprehensive proteomics sample preparation. Human megakaryoblastic leukaemia cancer cells’ protein extracts were treated in parallel with both Amicon-adapted eFASP and FASP, quantified for remaining SDS and then analyzed with a 1-hr gradient LC-MS/MS run. The Amicon-adapted eFASP utilizes a passivated low molecular weight cut-off Amicon filter, and incorporates a cleaning step with a high-content deoxycholate buffer and a ‘one-step-two-enzymes’ trypsin/Lys-C in-solution digestion. Amicon-adapted eFASP was found more reproducible and deepened proteome coverage, especially for membrane proteins. As compared to FASP, Amicon-adapted eFASP removed much of SDS from high-protein samples and reached a notable depth of proteome coverage with nearly 1,700 proteins identified in a 1 hr LC-MS/MS single-run analysis without prior fractionation. Amicon-adapted eFASP can therefore be regarded as a simple and reliable sample preparation approach for comprehensive proteomics.