Integrated Microfluidics for Protein Modification Discovery

Abstract

Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for highthroughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system's potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research. Molecular

Pathogen receptor discovery with a microfluidic human membrane protein array

“…These interactions point to a previously suggested common SV40 binding domain to TNR proteins (28). SV40 traffics via the endosomal pathway into the ER (29) and is known to bind to ganglioside GM1, present in caveolar/lipid raft domains at the plasma membrane (30,31). Notably, 23% (19 of 85) of our discovered interactions are located in lipid rafts, caveola, and endoplasmic reticulum (ER) membrane (SI Appendix, Table S2 and Dataset S2).…”

“…Membrane protein array generated by integrated microfluidic platform. (A) An integrated microfluidics platform (Left) was used for on-chip expression of membrane proteins, to serve as "baits" for protein interactions or modifications (29). The device consists of two polydimethylsiloxane (PDMS) layers, a flow layer with 64 × 64 unit cells array (gray), and a control layer with micromechanical valves (colored) that manipulate the flow of fluids in the experiment (Center).…”

A high-throughput integrated microfluidics method enables tyrosine autophosphorylation discovery

“…The shift from Tyr phosphorylation to Tyr autophosphorylation assay on our microfluidic platform 8 is conceptually simple (Fig. 1).…”

“…Interaction between proteins and nucleic acids was also showed 6 ,7 . More recently, the platform was proven to be compatible also with protein PTM analyses 8 . In that study, we applied recombinant enzymes or active cell extracts to the chip to promote PTM of fresh proteins in quasi-cellular environments.…”

“…Both the target protein and the protein modifier were then quantified colorimetrically to derive a normalized PTM signal. Tyrosine (Tyr) phosphorylation, ubiquitination, and ubiquitin chain preference was demonstrated 8 . Although functional as substrates for protein interactions and PTMs, it is still unclear whether the arrayed proteins maintain intrinsic catalytic activity.…”