Proteins are active cellular components that provide fundamental functions in living organisms, and their accurate measurement can yield essential knowledge to help understand and describe biological systems. Liquid chromatography-mass spectrometry (LC-MS) has emerged as the gold standard technique for high throughput identification and quantification of proteins in a large variety of samples.
Typically, proteomics sample preparation involves protein extraction from cells or tissues, followed by reduction and alkylation of disulfide bonds, then enzymatic digestion to shorter peptides that can be separated and detected by LC-MS analysis. Unfortunately, many of the most efficient extraction buffers considerably reduce proteolytic activity and/ or MS ionization efficiency, limiting overall proteome coverage. This means that they must be removed prior to protein digestion.
Single-pot, solid phase-enhanced sample preparation (SP3) technology was recently introduced as a rapid and efficient method for intact protein clean-up [1]. The SP3 protocol employs paramagnetic beads that are functionalized with hydrophilic groups for protein binding. This allows washing and digestion steps to be completed while proteins are bound to the beads. The workflow is compatible with a large variety of extraction buffers, leading to unbiased protein recovery with minimal sample processing. For improved sample quality with optimal digestion efficiency, the SP3- based protein clean-up can be readily combined with PreOmics’ robust iST technology for efficient protein alkylation, digestion and subsequent peptide clean-up. This novel SP3-iST workflow is highly versatile, and suitable for a wide variety of sample types.
This application note describes high throughput automation of the SP3-iST protocol on a Freedom EVO workstation, with automated peptide clean-up using the Resolvex A200 positive pressure processor. Starting from lysed and denatured samples, this automated workflow enables the processing of up to 96 samples (ie. cell lysates) in approximately five hours, while reducing overall hands-on time to less than one hour.
>> Download the Full Application Note as a PDF
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