One of the compelling needs in drug discovery is to connect cellular responses to pharmacological substance with that of drug mechanism of action (MOA). While genomic signatures provide an extensive view of genome-wide alterations, there are genome-independent mechanisms, such as posttranslational modifications (PTMs), that cannot be revealed through genomics. Besides, drugs that target cellular proteins which are interconnected and integrated with various biochemical pathways can be perturbed by those. Bruton"s tyrosine kinase inhibition (BTKi) shows therapeutic promise for the treatment of multiple sclerosis (MS). Potential mode of action that contribute to this efficacy includes the effects on both B lymphocytes and macrophage signaling cascades, as both cell types contribute to MS pathology and employ BTK signaling.

Towards better understand the biology of potential therapeutic mechanisms and to identify putative drug targets, it is advantageous to detect protein substrates that are phosphorylated by BTK in these cell-types under immune-responsive conditions. To attain a deeper proteome coverage and to avoid common issues such as low sensitivity, throughput, or reproducibility that are associated with conventional DDA-based mass spectrometry approaches, here we present label-free single-shot FAIMS-DIA mass spectrometry platform in exploring phospho-tyrosine networks of proteins downstream of BTK signaling, and subsequent validation by FAIMS-PRM mass spectrometry.

Human B lymphocytes (Ramos RA1 cell line) and THP-1-derived macrophages were immunologically stimulated by goat anti-human IgM F(ab")2 fragment and Fc OxyBURST, respectively, and with or without the presence of the BTK inhibitor tolebrutinib, and then perform tyrosine phosphoproteomics on the cell lysates. Together with ion-mobility and gas-phase ion separation at high resolution, the current phosphoproteomics platform can increase the sensitivity of detection, reducing chemical noises, improving the depth of phosphoprotein coverage and phospho-sites localization accurately. Besides, being label-independent, the FAIMS-DIA platform increases the throughput in sample processing and improves quantification, followed by multiplexed FAIMS-PRM for targeted assay.

Using tool compound targeting BTK (BTKi), we observed distinct downstream phospho-tyrosine signaling through FAIMS-DIA mass spectrometry in activated B cell and macrophages that highlights the organization of intrinsic cellular complexities of protein networks in achieving a desired pharmacological effect. A subset of phosphopeptides from these phospho-tyrosine networks has been further validated through targeted FAIMS-PRM and will be presented.

Comparison of tyrosine phosphoproteomics with and without BTKi in stimulated human B lymphocytes and macrophages will identify potential downstream targets of BTK in both cell types that will eventually provide strategies for an effective therapeutic development of MS.