Modular automated sample processing of biological samples using ultrasonication and a workstation for high-throughput proteomics

Sample preparation for mass spectrometry analysis involves numerous liquid transfer steps.

These include

• sample lysis,
• protein extraction,
• solubilisation,
• estimation,
• reduction and alkylation,
• normalisation,
• clean-up,
• enzymatic digestion,
• and desalting.

Adapting these steps onto an automated workstation can increase efficiency, throughput, and reduce coefficients of variance ( %CV ) thereby providing reliable reproducible data for statistical comparisons.

This protocol is part of a modular collection for the processing of biological samples for proteomics.

The entry point is ultrasonication of biological samples (cells, tissues, laser captured FFPE sections) using a plate based LE220+ system from Covaris, followed by sample processing with a Biomek NxP workstation.

Technical measurement of workflow percentage coefficients of variation (%CVs) using HeLa extract at the entry stage, to measurement of the ion intensities at the data processing stage with data collected by DDA using a QE-HF and processed using Progenesis-QI for proteomics has shown that one third of peptides have %CVs below 20% , and with 80% of peptides having %CVs below 30% . Using Progenesis QI for proteomics indicates that 98% of peptides are found in all 7 replicates processed.

In addition, the whole procedure (80 samples) may be completed in a day, if shorter digestion times are utilised.