License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: December 20, 2023
Last Modified: December 20, 2023
Collection Integer ID: 92551
Keywords: throughput proteomics sample preparation for mass spectrometry analysis, throughput proteomics sample preparation, proteomic, qi for proteomic, ultrasonication of biological sample, processing of biological sample, protein extraction, sample processing of biological sample, mass spectrometry analysis, progenesis qi for proteomic, biological sample, using ultrasonication, sample lysi, automated sample processing, sample processing, ultrasonication, biomek nxp workstation, using hela extract, hela extract at the entry stage, extraction, peptide, enzymatic digestion, using progenesis qi, using progenesi, data processing stage
Abstract
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.
Protein aggregation capture (PAC) and minimal automated processing for proteomics using magnetic beads and a Beckman Biomek™ NxP workstation for 96 well plates.
