Ultra Low-cost DNA Miniprep Spin-Columns for Rapid Filter-Trapped Proteomics Sample Preparation

Abstract

Complete proteolytic digestion in the preparation of proteins for bottom-up proteomic analysis is substantially improved by the use of detergents for complete denaturation. This however is incompatible with many proteases, and highly detrimental to LC-MS/MS data collection. Recently, filter-based methods such as FASP (Filter-Aided Sample Prep) have seen wide use due to their ability to remove detergents and other harmful reagents prior to digestion and mass spectrometric analysis. Unfortunately, these techniques can be variable and time consuming. Suspension trapping (S-Trapping) is a newer method that utilizes a depth-filter to trap flocculated proteins, and has proven to be a faster approach for proteomic analysis. Sample preparation by these methods requires careful control of protein concentrations in order to flocculate the sample for collection, and the cost of commercial solutions can be high. We hypothesized that protein suspensions also retain on silica-based filters due to ionic interactions mediated by the presence of sodium (Na+), SO42- and PO43-. As such, we sought to investigate if very low-cost DNA purification spin-filters, so called ‘minipreps’ could efficiently and reproducibly trap proteins for digest and LC-MS/MS analysis. Using model proteins and whole-cell lysates we compared digestion efficiencies, capacities, recovery and identification rates from samples prepared using DNA-minipreps and FASP-based protocols. Samples were analyzed using nano uHPLC MS-MS/MS and Label-Free-Quantitative (LFQ) proteomics. DNA-filters show low variability, excellent recovery, sensitivity, and proteome depth from a commercially obtainable device which costs < $0.25 (US) per sample.