Abstract
AbstractProteomic analysis by mass spectrometry (MS) of small (≤2 mg) solid tissue samples from diverse formats requires high throughput and comprehensive proteome coverage. We developed a near universal, rapid and robust protocol for sample preparation, suitable for high-throughput projects that encompass most cell or tissue types. This end-to-end workflow extends from original sample to loading the mass spectrometer and is centred on a one tube homogenisation and digestion method called Heat ‘n Beat (HnB). It is applicable to most tissues, regardless of how they were fixed or embedded. Sample preparation was divided to separate challenges. The initial sample washing, and final peptide clean-up steps were adapted to three tissue sources: fresh frozen (FF), optimal cutting temperature (OCT) compound embedded (FF-OCT), and formalin-fixed paraffin-embedded (FFPE). Thirdly, for core processing, tissue disruption and lysis were decreased to a 7 min heat and homogenisation treatment, and reduction, alkylation and proteolysis were optimised into a single step. The refinements produced near doubled peptide yield, delivered consistently high digestion efficiency of 85-90%, and required only 38 minutes for core processing in a single tube, with total processing time being 53-63 minutes. The robustness of HnB was demonstrated on six organ types, a cell line and a cancer biopsy. Its suitability for high throughput applications was demonstrated on a set of 1,171 FF-OCT human cancer biopsies, which were processed for end-to-end completion in 92 hours, producing highly consistent peptide yield and quality for over 3,513 MS runs.Graphical Abstract
Publisher
Cold Spring Harbor Laboratory