Advancing Electrophoretic Assays for Protein Analysis through Mass Spectrometry Imaging, Microfluidic Methods, and Hydrogel Engineering

Proteoforms, highly related proteins, play critical roles in disease processes such as cancer. However, studying proteoforms is challenging due to their high homology and low abundance. Electrophoretic assays are capable of elucidating proteoforms, which are often indistinguishable with canonical antibody-based protein assays and mass spectrometry. This dissertation presents advancements in microfluidic, material, and mass spectrometry imaging approaches towards improved multiplexing, proteoform specificity, and sensitivity of electrophoretic assays./nFirst, the integration of mass spectrometry imaging and proteoform separation tools paves the way for highly multiplexed single-cell proteomics. Single-cell immunoblotting (scIB) offers proteoform detection specificity but often relies on fluorescence-based readout, limiting its multiplexing capability. Among the rising multiplexed imaging methods is multiplexed ion beam imaging by time-of-flight (MIBI-TOF), a mass spectrometry imaging technology. MIBI-TOF employs metal-tagged antibodies that do not suffer from spectral overlap to the same degree as fluorophore-tagged antibodies. We report for the first time MIBI-TOF of single-cell immunoblotting (scIB-MIBI-TOF). The scIB assay subjects single-cell lysate to protein immunoblotting on a microscale device consisting of a 50- to 75-µm thick hydrated polyacrylamide (PA) gel matrix for protein immobilization before in-gel immunoprobing. We confirm antibody-protein binding in the PA gel with indirect fluorescence readout of metal-tagged antibodies. Since MIBI-TOF is a layer-by-layer imaging technique, and our protein target is immobilized within a 3D PA gel layer, we characterize the protein distribution throughout the PA gel depth by fluorescence confocal microscopy and confirm that the highest signal-to-noise ratio is achieved by imaging the entirety of the PA gel depth. Accordingly, we report the required MIBI-TOF ion dose strength needed to image varying PA gel depths. Lastly, by imaging ∼42% of the PA gel depth with MIBI-TOF, we detect two isoelectrically separated TurboGFP (tGFP) proteoforms from individual glioblastoma cells, demonstrating that highly multiplexed mass spectrometry-based readout is compatible with scIB./nNext, a polydimethylsiloxane (PDMS)-based device miniaturizes the isoelectric focusing (IEF) assay, enabling the specific measurement of proteoforms on a chip the size of a microscope slide. Microfluidic IEF assays have made it possible to assay proteoforms from low starting cell numbers, yet these often rely on carrier ampholytes (CAs) to establish a pH gradient for protein separation, and CAs have major limitations, including lack of pH gradient tunability and stability. Immobilized pH gradient (IPG) gels have been developed to overcome these limitations, but efforts to implement IPGs at the microscale have been limited to difficult-to-manufacture glass devices and require proteins to be labeled before analysis, precluding complex samples such as cell lysate. Here, we introduce the first PDMS-based IPG microfluidic device (µIPG). First, we establish a pH gradient by introducing acidic and basic gel precursors at two reservoirs flanking a separation channel and allow diffusion to establish a linear pH gradient within the separation channel. We introduce a 2-step photopolymerization procedure to create a composite gel with two functions: 1) IEF protein separation via the IPG gel component, and 2) protein capture for downstream immunoprobing via a photoactive gel component. Therefore, µIPG is suitable for the analysis of unlabeled, complex samples, which we demonstrate by immunoprobing green fluorescent protein (GFP) from GFP-expressing breast cancer cells. Moreover, we show that the pH gradient in the PDMS-based µIPG is stable for at least 30 minutes, and we are able to resolve proteoforms differing by about 0.1 isoelectric point./nFurthermore, we continue developing gradient hydrogel technology to engineer a pore-size gradient gel for single-cell 3D projection electrophoresis. Single-cell 3D projection electrophoresis was developed by our group to increase throughput over planar (2D) single-cell western blotting and to provide the option to preserve spatial context in the analysis of intact tissue. However, it can be difficult to achieve adequate protein separation in the short separation distance (1 mm) employed by 3D projection electrophoresis. Conventional western blots have employed a gradient pore size in the axis of separation to improve separation efficiency. We describe the development of a gel slab for 3D projection electrophoresis with a gradient pore-size in the z-direction (axis of separation)./nLastly, an enclosed single-cell electrophoretic assay for native electrophoresis is developed. Single-cell electrophoresis (scEP) is a powerful method for separating proteoforms based on size and charge within individual cells. However, the current implementation of scEP is limited to open microfluidic devices, which precludes its integration with enclosed microfluidic formats (e.g., for microchannel electrophoresis), which could introduce additional performance improvements and separation modalities to scEP. We present an enclosed microfluidic scEP device that combines hydrodynamic cell trapping, electrical lysis, and protein electrophoresis for the first time. The device incorporates a PDMS-hydrogel hydrodynamic cell trapping component that also functions as an electrical lysis component. With electrical cell lysis, rapid (<1 second) and non-denaturing lysis of proteins is achieved, preserving their native state during both lysis and electrophoresis. The microfluidic scEP device we introduce here demonstrates the feasibility and challenges of employing an enclosed microfluidic design for scEP./nBy integrating diverse electrophoretic and mass spectrometry imaging tools in a miniaturized format, this dissertation expands the toolkit for proteoform analysis.

Advisor: Herr, Amy E