Modern mass spectrometry (MS)-based phosphoproteomics quantifies thousands of phosphorylation sites. However, most of them have no experimentally derived biological function or cognate kinase. A promising strategy to unravel phosphorylation networks in more detail is to study their dynamic response to multiple internal or external perturbations in a systematic manner. To facilitate such high-dimensional experimental designs, we recently introduced µPhos, a scalable and sensitive platform for phosphopeptide enrichment (Oliinyk et al. 2024). By minimizing processing volumes and transfer steps, µPhos achieves lossless processing of 96-well cell culture plates or other input-limited samples within one day. Combined with trapped ion mobility – mass spectrometry, we quantified >10,000 class I phosphosites with excellent reproducibility (median CV < 15%) and data completeness (>99%) from 10 µg protein starting amount, which equates the protein mass of ~40,000 human cancer cells cultured in a 96-well format. Diluting the input amount to 250 ng and 62.5 ng, we still identified about 2,000 and 700 phosphosites with a localization probability >0.75. Considering that many emerging proteomics applications demand such or even lower input amounts, we here explored strategies to further miniaturise our setup. Specifically, we tested non-isobaric labelling-based multiplexed data-independent acquisition to simultaneously increase throughput and sensitivity.
Pilot experiments indicated that 96-well plates are not ideal for phosphopeptide enrichment from low (<1µg) protein starting inputs. We reasoned that this is because of the volume-to-area ratio in 96-well plates, which lowers the concentration of peptides and results in adsorptive losses at plastic surfaces. To solve this problem, we explored a combination of 384-well plate sample preparation and stable isotope labelled dimethyl-based multiplexing. Depending on the combination of labels and digestion enzyme, this should offer an up to five-fold increase in sensitivity and sample throughput. To make µPhos compatible with dimethyl labelling, we optimized cell lysis and alkylation/reduction buffers to achieve a high labelling efficiency (>95%) of tryptic peptides. To evaluate the quantitative accuracy with multiplexed diaPASEF, we performed benchmarking experiments of samples mixed in known ratios and also test the influence of gradient length and MS instrumentation for different input amounts in the sub-µg range. Collectively, these results demonstrate the feasibility of high-dimensional phosphoproteomics experiments, for example to screen drug response in a time- and dose-dependent manner.