Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

Abstract

This protocol describes a highly reproducible antibody-based method providing protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV activated linking chemistry to detect changes in phosphorylation status. We describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adapter protein CrkL a major substrate of the oncogenic tyrosine kinase BCR/ABL in chronic myeloid leukaemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5pg/nL limit of protein detection (<0.2% of a stem cell sample containing <10 4 cells). Additional assays are described for pTyr207-CrkL and PTPRC/CD45, developed using this protocol and applied to CML patient samples. This method is high throughput and can act as a screen for in vitro cancer stem cell response to drugs and novel agents. SummaryThis protocol uses a highly sensitive and reproducible antibody-based capillary isoelectric focusing approach to establish protein phosphorylation status from nanogram quantities of protein cell lysate from cell line or primary stem cell material. Is-protocol-to

Capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics

“…Nevertheless, when well-validated antibodies are available, charge-CNIA assays are a uniquely powerful tool for generating information that is cumbersome or impossible to generate using other approaches. Antibodies developed for Human Protein Atlas project (http://www.proteinatlas.org/) has been reported may serve as a good source for charge-CNIA assay development [42]. …”

“…Nanogram level sample consumption, digital data processing and quantitation, and proven assay reproducibility make the CNIA system appealing for analysis of clinical specimens to facilitate assessment of pharmacodynamic biomarkers and treatment evaluation. The technology was demonstrated to allow the measurement of stem cell signals from a total of <10,000 cells, which contains a stem cell population of <0.2% [42]. …”

Fast-Acting and Receptor-Mediated Regulation of Neuronal Signaling Pathways by Copaiba Essential Oil

“…Future integration of nanofluidic protein PTM profiling and chemical composition analysis could improve the quality control of copaiba essential oil by identifying permissible chemical variations that do not affect its biological activities. The proven capabilities of nanofluidic protein PTM profiling technology, including its ultrasensitivity, high reproducibility, high throughput and automatic operation [14,15], will be invaluable for preclinical quality testing and clinical evaluation of the pharmacodynamics of copaiba essential oil.…”

“…The effects of copaiba essential oil on SH-SY5Y cells were characterized using nanofluidic protein post-translational modification (PTM) profiling technologies, which measure changes in the PTM profiles of signaling proteins [13,14]. Briefly, total protein extracts of SH-SY5Y cells were separated by charge or size using capillary isoelectric focusing (cIEF) or Western immunoassays in matrix-filled capillaries, respectively [15]. Changes in the isoelectric points or in the binding of primary antibodies that recognize specific modification sites were used to measure protein PTMs.…”

Use of Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes (hiPSC‐CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways

“…Proteins and their respective phosphorylated isoforms are separated by charge in this nanofluidic proteomic immunoassay (NIA) that enables multiple protein phosphorylation isoforms to be resolved and detected simultaneously with the non-phosphorylated protein using the same primary antibody. NanoPro™ Simple Western (ProteinSimple) is an automated capillary isoelectric focusing immunoassay in which proteins and their respective isoforms (e.g., phosphorylated forms) are separated by charge, followed by target-specific antibody probing (Aspinall-O'Dea et al, 2015; Bradley et al, 2014; Chen et al, 2013b; Eldridge et al, 2014; Fan et al, 2009; O'Neill et al, 2006). Thus, NanoPro™ can simultaneously separate, detect and quantify multiple protein phosphorylation isoforms allowing a more sensitive and accurate analysis of cell signaling events.…”