Proteoform analysis has been recognized as a critical step in understanding the pathological mechanisms in disease formation and progression. Despite this, most analytical techniques fail to accurately record changes in the proteome such as Post Translational Modification (PTM) abundance changes, and products of proteolytic cleavage. Current efforts such as PTM enrichment kits used to analyse these characteristics, are largely targeted at specific modifications and as such are only suitable for specific use cases. With this in mind, there is a clear need for an analytical technique that is able to provide a form of proteome analysis that analyses PTMs and products of proteolytic cleavage in an untargeted manner.
In this study we aimed to use 2D-PAGE as a form of physical and chemical separation for total proteome screening of PTM’s. The two dimensions of separation (mass and isoelectric point) allow a significant reduction in sample complexity while also concentrating like proteoforms together. Samples were prepared Using UTC7 to extract proteins then reduced and alkylated with Tributylphosphine and acrylamide monomers. This was then followed by acetone precipitation, IEF and 2D-PAGE. Gels were stained with Coomassie, rapid destained and then “press cut” into square 96 deep welled plates. In gel digestion was then performed for all gel pieces in parallel followed by STAGE tipping in an apparatus that allows 96 samples to be STAGE tipped simultaneously.
Pilot data has presented this technique as capable of separating a proteome into 88 identical gel cubes that then translate into 88 samples for mass spectrometry analysis. Preliminary data has shown that this methodology has the ability to identify proteoform shifts such as PTM abundance changes and the presence of proteolytic cleavage. To counter the large number of samples, these are analysed through a short mass spectrometry gradient allowing an entire gel to go through a mass spectrometer in 24 hours. Paired with the workflow for rapid slicing and STAGE tipping this allows what previously would have taken months to be done in less than a day
Based on these findings, our future directions are the optimisation of a gradient to maximise analysis speed and sensitivity, determining optimal protein quantity for IEF and determining the impact of using reagents such as DNases and protease inhibitors
The optimisation of these steps will therefore provide whole gel proteoform data with only a few hours of human handling time and less than two days instrument time.