Rapid and Robust Bottom-up Sample Preparation in the ProTrap XG

Jessica L. Nickerson, Alan A. Doucette
Department of Chemistry, Dalhousie University; Halifax, Canada

Introduction

The ProTrap XG is a two stage spin cartridge that was developed in our lab to facilitate solvent precipitation based proteome workflows [1]. We have since developed an acetone precipitation protocol which achieves quantitative proteome recovery in minutes [2].

Following precipitation, conventional digestion procedures rely on an overnight digest, which limits the overall preparative throughput. High temperature and the addition of calcium ions have been reported to accelerate digestion [3 4 5] however, these conclusions are often based on abundant MS identifications, even though more identifications correlate with a less complete digestion. We herein present a 1 hour trypsin digestion that exploits the high activity seen at elevated temperatures in combination with the preservation effects of calcium ions, showing equivalent digestion completion to a conventional overnight digest.