Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Heat and Beat: A one-pot rapid tissue sample preparation for proteomics in under an hour (#194)

Dylan Xavier 1 , Clare Loudon 1 , Peter G Hains 1 , Phil J Robinson 1
  1. ProCan, CMRI, Westmead, NSW, Australia

Clinical proteomics on solid tissue samples from human tumour biopsies present multiple challenges. These include limited sample availability, reducing endogenous protease activity and challenges with efficiently solubilising and digesting proteins for high throughput mass spectrometry while achieving comprehensive proteome coverage. Protocols for cancer biopsy proteomics involves three sample preparation steps: tissue washing, lysis and digestion, followed by peptide clean-up by SPE. Our team have reported improvements in all three steps that greatly reduce processing time, enabling greater automation and higher sample throughput.

Our aim was to develop a universal protocol for tissue sample preparation that is quick and simple while improving protein solubilization, proteome coverage and reproducibility. Sample preparation with small (<2 mg) biopsies pose additional limitations including the risk of sample loss and introducing variability, partly due to multiple reagent addition/removal steps. We now report a one-pot method called heat and beat (HnB).

By identifying limitations and challenges of existing protocols, we developed a new protocol with fewer steps by achieving lysis, reduction, alkylation and digestion in essentially a single step which was preceded by a 7 min sample heat treatment step. This was achieved by several changes to the existing protocol including the combined use of a beadbeater for tissue homogenisation followed by pressure cycling technology (PCT) using a Barocycler to further lyse and digest the sample. The procedure requires 38 minutes and was tested on 6 tissue types, across three tissue embedding techniques, fresh frozen, OCT embedded (FF-OCT), and FFPE.

Our results show that HnB is an efficient new one-pot procedure. The protocol significantly reduced endogenous protease activity, improves peptide yield by up to 3-fold and delivers a digestion efficiency of 85-90% from each sample source. We obtained an increase in the number of proteins identified of up to two-fold while reducing variability and processing time.