Microfluidic Approaches for Probing Protein Phosphorylation in Cells

Protein phosphorylation plays critical roles in diverse cellular functions, including cell cycle, growth, differentiation, and apoptosis. Deregulated phospho-signaling is often associated with many human diseases and cancers. Despite tremendous efforts to investigate the molecular mechanisms that control the functionality of phospho-signaling pathways, only limited progress has been made on describing the temporal and spatial profiles of cellular protein phosphorylation. The main challenges associated with the study of phospho-signaling processes in cells are related to the short time-scale of certain phosphorylation and dephosphorylation events, the low abundance of the phosphorylated protein forms as compared to their non-phosphorylated counterparts, the complicated and time-consuming sample preparation methods that are accompanying such type of work, and, last, the performance of the detection methods that are suitable for assessing protein phosphorylation. To tackle the challenges associated with the investigation of protein phosphorylation in cells, our objective was to develop a combined mass spectrometry (MS) and microfluidics strategy that enables fast sampling and sensitive detection of key signaling phosphoproteins in complex biological samples. MS is the most widely used analytical tool in the field of proteomics due to its high sensitivity, specificity, and throughput. Microfluidics has been proven as a suitable platform for handling small volumes of scarce samples, being also amenable to automation, integration, and multiplexing. To achieve our objective, this study was conducted in multiple steps: (1) We performed a comprehensive analysis of the factors that affect the performance of mass spectrometry detection (i.e., sensitivity, reproducibility, ability to accurately identify a large number of proteins from complex samples), when used in conjunction with technologies that are conducted in a non-standard fashion, on short time-scales; (2) We developed and evaluated a miniaturized strategy for rapid proteolytic digestion and phosphopeptide enrichment; (3) We demonstrated sensitive detection and quantification of phosphopeptides from complex biological samples using multiple reaction monitoring mass spectrometry (MRM-MS) and microfluidic sample processing; and (4) We developed a microfluidic platform for handling and processing cells that enables the investigation of biological processes that occur on short time-scales, and that can be integrated with the devices developed for the analysis of phospho-proteins. SKBR3 cells were used as a model system for developing and demonstrating the microfluidic chips. The detection and quantification of phospho-proteins involved in MAPK (mitogen activated protein kinase) signaling pathways was achieved at the low nM level. Overall, this study demonstrates proof-of-concept applicability of a microfluidics-MS strategy for monitoring phosphorylation processes in signaling networks.