Digital Receptor Occupancy Assay in Quantifying On- and Off-Target Binding Affinities of Therapeutic Antibodies.

While monoclonal antibodies are the fastest-growing class of therapeutic agents, we lack a method that can directly quantify the on- and off-target binding affinities of newly developed therapeutic antibodies in crude cell lysates. As a result, some therapeutic antibody candidates could have a moderate on-target binding affinity but a high off-target binding affinity, which not only gives a reduced efficacy but triggers unwanted side effects. Here, we report a single-molecule counting method that precisely quantifies antibody-bound receptors, free receptors, and unbound antibodies in crude cell lysates, termed digital receptor occupancy assay (DRO). Compared to the traditional flow cytometry-based binding assay, DRO assay enables direct and digital quantification of the three molecular species in solution without the additional antibodies for competitive binding. When characterizing the therapeutic antibody, cetuximab, using DRO assay, we found the on-target binding ratio to be 65% and the binding constant (Kd) to be 2.4 nM, while the off-target binding causes the binding constant to decrease by 0.3 nM. Other than cultured cells, the DRO assay can be performed on tumor mouse xenograft models. Thus, DRO is a simple and highly quantitative method for cell-based antibody binding analysis which can be broadly applied to screen and validate new therapeutic antibodies.

A flow-proteometric platform for analyzing protein concentration (FAP): Proof of concept for quantification of PD-L1 protein in cells and tissues.

Protein expression level is critically related to the cell physiological function. However, current methodologies such as Western blot (WB) and Immunohistochemistry (IHC) in analyzing the protein level are rather semi-quantitative and without the information of actual protein concentration. We have developed a microfluidic technique termed a "flow-proteometric platform for analyzing protein concentration (FAP)" that can measure the concentration of a target protein in cells or tissues without the requirement of a calibration standard, e.g., the purified target molecules. To validate our method, we tested a number of control samples with known target protein concentrations and showed that the FAP measurement resulted in concentrations that well matched the actual concentrations in the control samples (coefficient of determination [R2], 0.998), demonstrating a dynamic range of concentrations from 0.13 to 130 pM of a detection for 2 min. We successfully determined a biomarker protein (for predicting the treatment response of cancer immune check-point therapy) PD-L1 concentration in cancer cell lines (HeLa PD-L1 and MDA-MB-231) and breast cancer patient tumor tissues without any prior process of sample purification and standard line construction. Therefore, FAP is a simple, faster, and reliable method to measure the protein concentration in cells and tissues, which can support the conventional methods such as WB and IHC to determine the actual protein level.

Analysis of Individual Signaling Complexes by mMAPS, a Flow-Proteometric System.

Signal transduction is essential for maintaining normal cell physiological functions, and deregulation of signaling can lead to diseases such as diabetes and cancers. Some of the major players in signal delivery are molecular complexes composed of proteins and nucleic acids. This unit describes a technique called microchannel for multiparameter analysis of proteins in a single complex (mMAPS) for analyzing and quantifying individual target signaling complexes. mMAPS is a flow-proteometric system that allows detection of individual proteins or complexes flowing through a microfluidic channel. Specific target proteins and nucleic acids labeled by fluorescent tags are harvested from tissues or cultured cells for analysis by the mMAPS system. Overall, mMAPS enables both detection of multiple components within a single complex and direct quantification of different populations of molecular complexes in one setting in a short timeframe and requiring very low sample input.

PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response.

Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment.

mMAPS: a flow-proteometric technique to analyze protein-protein interactions in individual signaling complexes.

Signal transduction is a dynamic process that regulates cellular functions through multiple types of biomolecular interactions, such as the interactions between proteins and between proteins and nucleic acids. However, the techniques currently available for identifying protein-protein or protein-nucleic acid complexes typically provide information about the overall population of signaling complexes in a sample instead of information about the individual signaling complexes therein. We developed a technique called "microchannel for multiparameter analysis of proteins in a single complex" (mMAPS) that simultaneously detected individual target proteins either singly or in a multicomponent complex in cell or tissue lysates. We detected the target proteins labeled with fluorophores by flow proteometry, which provided quantified data in the form of multidimensional fluorescence plots. Using mMAPS, we quantified individual complexes of epidermal growth factor (EGF) with its receptor EGFR, EGFR with signal transducer and activator of transcription 3 (STAT3), and STAT3 with the acetylase p300 and DNA in lysates from cultured cells with and without treatment with EGF, as well as in lysates from tumor xenograft tissue. Consistent with the ability of this method to reveal the dynamics of signaling protein interactions, we observed that cells treated with EGF induced the interaction of EGF with EGFR and the autophosphorylation of EGFR, but this interaction decreased with longer treatment time. Thus, we expect that this technique may reveal new aspects of molecular interaction dynamics.

Microfluidic three-dimensional hydrodynamic flow focusing for the rapid protein concentration analysis.

A simple microfluidic 3D hydrodynamic flow focusing device has been developed and demonstrated quantitative determinations of quantum dot 525 with antibody (QD525-antibody) and hemagglutinin epitope tagged MAX (HA-MAX) protein concentrations. This device had a step depth cross junction structure at a hydrodynamic flow focusing point at which the analyte stream was flowed into a main detection channel and pinched not only horizontally but also vertically by two sheath streams. As a result, a triangular cross-sectional flow profile of the analyte stream was formed and the laser was focused on the top of the triangular shaped analyte stream. Since the detection volume was smaller than the radius of laser spot, a photon burst histogram showed Gaussian distribution, which was necessary for the quantitative analysis of protein concentration. By using this approach, a linear concentration curve of QD525-antibody down to 10 pM was demonstrated. In addition, the concentration of HA-MAX protein in HEK293 cell lysate was determined as 0.283 ± 0.015 nM. This approach requires for only 1 min determining protein concentration. As the best of our knowledge, this is the first time to determinate protein concentration by using single molecule detection techniques.

Rapid detection of two-protein interaction with a single fluorophore by using a microfluidic device.

We have developed a microfluidics based platform and methodology named MAPS (microfluidic system for analyzing proteins in single complex) for detecting two protein interactions rapidly using a single fluorophore. Target proteins were labelled with Quantum dot 525 (QD525) via specific polyclonal antibodies, and were transported through the microfluidic channel subsequently, where the 375 nm excitation laser light was focused to form a detection volume. Photon bursts from target proteins passing through the detection volume were recorded and their photon burst histograms were plotted which demonstrated roughly the specific protein interaction ratio based on their population and statistical behavior. As a proof of concept, Src/STAT3 protein complex interaction ratios with and without EGF stimulation were obtained by MAPS within 1 h and the results were well matched with the one obtained by the conventional immunoprecipitation/Western blot (IP/WB).

Rapid antibiotic efficacy screening with aluminum oxide nanoporous membrane filter-chip and optical detection system.

We have developed a filter-chip and optical detection system for rapid antibiotic efficacy screening. The filter-chip consisted of a 1-mL reservoir and an anodic aluminum oxide (AAO) nanoporous membrane. Sample solution with liquid growth media, bacteria, and antibiotics was incubated in the reservoir for a specific period of time. The number of live bacteria on the surface of membrane was counted after the incubation with antibiotics and filtration. Using this biosensing system, we have demonstrated a 1-h antibiotic screening for patients' clinical samples, significantly faster than the conventional antibiotic susceptibility tests that typically take more than 24h. This rapid screening nature makes the filter-chip and detection system ideal for tailoring antibiotic treatment to individual patients by reducing the microbial antibiotic resistance, and improving the survival rate for patients suffering from postoperative infections.

High speed digital protein interaction analysis using microfluidic single molecule detection system.

The understanding of protein interaction dynamics is important for signal transduction research but current available techniques prove difficult in addressing this issue. Thus, using the microfluidic approach, we developed a digital protein analytical platform and methodology named MAPS (Microfluidic system Analyzing Protein in Single complex) that can measure the amount of target proteins and protein complexes at the digitally single molecule resolution. By counting protein events individually, this system can provide rough protein interaction ratios which will be critical for understanding signal transduction dynamics. In addition, this system only requires less than an hour to characterize the target protein sample, which is much quicker than conventional approaches. As a proof of concept, we have determined the interaction ratios of oncogenic signaling protein complexes EGFR/Src and EGFR/STAT3 before and after EGF ligand stimulation. To the best of our knowledge, this is the first time that the interaction ratio between EGFR and its downstream proteins has been characterized. The information from MAPS will be critical for the study of protein signal transduction quantitation and dynamics.

MEASUREMENT of Protein 53 Diffusion Coefficient in Live HeLa Cells Using Raster Image Correlation Spectroscopy (RICS).

We have applied Raster Image Correlation Spectroscopy (RICS) technique to characterize the dynamics of protein 53 (p53) in living cells before and after the treatment with DNA damaging agents. HeLa cells expressing Green Fluorescent Protein (GFP) tagged p53 were incubated with and without DNA damaging agents, cisplatin or eptoposide, which are widely used as chemotherapeutic drugs. Then, the diffusion coefficient of GFP-p53 was determined by RICS and it was significantly reduced after the drug treatment while that of the one without drug treatment was not. It is suggested that the drugs induced the interaction of p53 with either other proteins or DNA. Together, our results demonstrated that RICS is able to detect the protein dynamics which may be associated with protein-protein or protein-DNA interactions in living cells and it may be useful for the drug screening.