Combining Proteome Integral Solubility Alteration (PISA) assay and one-tip workflow for a streamlined and high-throughput drug target deconvolution

The Proteome Integral Solubility Alteration (PISA) assay is a well-established mass spectrometry (MS)-based proteomics methodology. It identifies changes in protein-ligand complexes based on their solubility variation under different treatment conditions based on thermal probing at several temperatures and is applicable to living cells and cell lysates. PISA usually necessitates relatively high sample input, but biomedically-relevant material available for analysis is often limited. This is the case for patient derived samples or challenging and costly cell cultures, and thus reducing sample requirements would increase the widespread applicability of PISA. Utilizing the One-Tip workflow, a novel sample preparation technique based on the Evotip, streamlines analysis of low sample input due to direct protein digestion on top of the C18-disk in the tip, followed by desalting, concentration and injection into the Evosep One LC system.

The addition of 0.2% n-Dodecyl-Beta-Maltoside (DDM) during the freeze-thaw cell lysis after the PISA assay allows for a swift transition to the One-Tip method. Since DDM is a MS compatible detergent, there is no need to perform a buffer exchange of the protein sample prior to enzymatic Lys-C/trypsin digestion, thus streamlining the method by decreasing sample preparation time as well as minimizing variability thanks to fewer pipetting steps. To test the sensitivity of the integration of PISA with the One-Tip workflow, we assayed the cellular targets of staurosporine, a broadband kinase inhibitor, in different quantities of HeLa cells. The PISA samples were analyzed on the Orbitrap Astral mass spectrometer using narrow-window DIA. As a readout, we investigated the correlation between cell number input and subsequent protein IDs and identified kinase targets. This showed that One-Tip-PISA can be performed on very low cell inputs, as low as 2500 HeLa cells for each temperature point sampled. This yielded reproducible results showing a strong correlation with known staurosporine targets, surpassing the protein depth and sample reproducibility of comparable methods like the Protein Aggregation Capture (PAC) protocol, when using a limited sample amount. Furthermore, the One-Tip-PISA method enabled us to prepare and analyze a full 96-well plate of HeLa cells treated with 11 different kinase inhibitors at 2 different concentrations within a single day. The resulting injection-ready Evotips can be analyzed within a subsequent day by fast LC-gradients. The One-Tip-PISA method is promising for higher throughput and lower sample input compared to the existing PISA analysis, with potential applications in a wide range of research areas.