Poster Presentation 24th Annual Lorne Proteomics Symposium 2019

EasyPep - A new simplified and optimized workflow for MS sample preparation (#108)

Michael MM Mariani 1 , Amarjeet Flora 2 , Sergei Snovida 2 , Ryan Bomgarden 2
  1. ThermoFisher Scientific, Melbourne, VIC, Australia
  2. Thermo Fisher Scientific, Rockford, IL, USA

Advances in mass spectrometry has enabled routine analysis of complex protein samples. However, sample preparation methods are not standardized with many protocols taking 8-24hrs in addition to suffering from low peptide yields, digestion efficiency and reproducibility. Here, we describe a simplified sample prep kit containing pre-formulated reagents and standardized protocol that can be used to efficiently process 10µg to 100µg protein samples in less than 2 hours. In this study, we evaluated the scalability, compatibility, and reproducibility of this sample preparation kit compared to published methods.
Protein extraction from cells and tissues was evaluated using a cell lysis buffer and universal nuclease for nucleic acid disruption compared to standard sonication methods. A rapid (<10min.) reduction/alkylation solution was developed as well as a combined trypsin/LysC protease mixture for protein digestion.  A mixed mode peptide clean-up procedure using a novel spin column format was used for detergent removal. Peptides were quantified and normalized using the PierceTM Quantitative Colorimetric Peptide Assay prior to analysis using a Thermo Scientific™ Q Exactive™ HF MS.
Our new standardized workflow yielded 10-20% higher number of peptides and proteins with lower missed cleavages (<90%) compared to other commercial kits or homebrew methods. We demonstrate that our optimized protocol reduces hands on time to less than 30 minutes with total sample processing time from intact cells to cleaned-up peptides under 1.5 hours.  Finally, we show this procedure is compatible with isobaric labeling reagents such as TMT and LFQ methods to reproducibly quantify protein abundances. The protocol has been successfully tested with several cell lines (HeLa, CHO, A549, 293), purified proteins and various mouse tissues (heart, liver, lung).
Overall, our kit provides superior method in terms of time saved, peptide/protein identification rates, and reproducibility compared to previous proteomic methods and greatly simplifies proteomic sample preparation for protein identification and quantitation.