Existing cytokine assay formats are currently the gold standard for analysing secreted proteins from a given system. These assays utilise secondary antibody read-outs and are very sensitive, however they are limited in terms of depth, incredibly costly (thousands of pounds per plate) and are unable to perform an un-targeted global analysis of a systems secretome. Advances in MS-based proteomics sample preparation and technology make it an advantageous platform for studying the secretome at pace. In addition, an MS approach allows both un-targeted global analyses and targeted PRM analyses from a single sample. The aim of this work was to develop and apply a high-throughput compatible workflow for profiling the human secretome by MS-based proteomics.
All experimental analysis was performed on an EvosepOne liquid chromatography system (Evosep Biosystems) coupled with the timsTOF Pro2 mass spectrometer (Bruker Daltonics). In the first step of prm-PASEF acquisition method development, a panel of cytokines, chemokines and growth factors were digested using Trypsin and analysed on the 100 SPD (11 minutes) LC gradient using standard DDA-PASEF acquisition. To create an initial prm-PASEF target list, a database search was conducted in FragPipe before filtering to remove non-unique peptides and those with missed cleavages or methionine oxidation.
The initial prm-PASEF target list was tested using individual cytokine standards and the corresponding transition list was generated using Skyline software. Here, the top performing peptides for each protein were selected and collated into the final prm-PASEF target list. The linear detection range for each peptide was subsequently determined in prm-PASEF mode on the 100, 200 and 300 SPD (11, 5 and 3 minutes, respectively) LC gradients. Quantitation was performed using peptide standard curves spanning a 1000-fold concentration range over eleven concentration levels in both mobile phase and in a surrogate extracellular matrix.
Finally, as a proof-of-concept study, we treated human iPSC derived macrophages with various inflammatory stimuli before harvesting cell pellets and supernatants for both global and targeted proteomic analysis. Here, we were able to demonstrate that this workflow can be used for discriminating between different inflammatory phenotypes. A parallel study was also set up in the same cell type, stimulating samples with various Toll-like receptor agonists before harvesting cell pellets and supernatants over 24 hours for proteomic analyses; enabling the generation of a kinetic profile of cytokine release over this time period.