Ultrasensitive proteomic quantitation of cellular signaling by digitized nanoparticle-protein counting.

Thomas Jacob,Damien Ramunno-Johnson,Thomas O’Hare,Tania Q Vu,Anupriya Agarwal,Jeffrey W Tyner,Mehmet Gönen,Brian J Druker

doi:10.1038/srep28163

Abstract

Many important signaling and regulatory proteins are expressed at low abundance and are difficult to measure in single cells. We report a molecular imaging approach to quantitate protein levels by digitized, discrete counting of nanoparticle-tagged proteins. Digitized protein counting provides ultrasensitive molecular detection of proteins in single cells that surpasses conventional methods of quantitating total diffuse fluorescence, and offers a substantial improvement in protein quantitation. We implement this digitized proteomic approach in an integrated imaging platform, the single cell-quantum dot platform (SC-QDP), to execute sensitive single cell phosphoquantitation in response to multiple drug treatment conditions and using limited primary patient material. The SC-QDP: 1) identified pAKT and pERK phospho-heterogeneity and insensitivity in individual leukemia cells treated with a multi-drug panel of FDA-approved kinase inhibitors, and 2) revealed subpopulations of drug-insensitive CD34+ stem cells with high pCRKL and pSTAT5 signaling in chronic myeloid leukemia patient blood samples. This ultrasensitive digitized protein detection approach is valuable for uncovering subtle but important differences in signaling, drug insensitivity, and other key cellular processes amongst single cells.

Highlights

We demonstrate that the Single cell quantum-dot platform (SC-QDP) quantitates phosphoresponse heterogeneity in human acute myeloid leukemia MOLM14 cells to kinase inhibitor drugs (KIs) and identifies KI-insensitive CD34+cells in patients diagnosed with chronic myeloid leukemia
Given that many proteins of interest and disease importance, such as signaling and regulatory proteins, are present at low abundance in single cells[3,4,5,26], and that such proteins can be further reduced by treatment with therapeutic compounds, this digitized molecular counting approach is of broad value for the quantitative study of major cellular signaling processes, as well as to detect other molecules of expected low abundance in single or rare populations of cells
The value of the high sensitivity achieved by digitized proteomic quantitation is illustrated by our implementation of an integrated SC-QDP platform to measure phosphoprotein signaling in individual leukemia cells

Summary

Introduction

QD are intensely bright, bleaching-resistant semiconductor nanoparticles that have matured as valuable probes for multi-color immunofluorescence and for tracking the dynamics of single molecules[22,23] yet, the advantages of digitized proteomic quantification using QDs, or other dyes have not been fully recognized. The SC-QDP has very high cell retention, enabling assays of limited quantities of cell sample, thereby overcoming a major bottleneck in assay of primary patient material. We demonstrate that the SC-QDP quantitates phosphoresponse heterogeneity in human acute myeloid leukemia MOLM14 cells to kinase inhibitor drugs (KIs) and identifies KI-insensitive CD34+cells in patients diagnosed with chronic myeloid leukemia. The molecular sensitivity offered by this digitized proteomic approach is valuable for revealing differences in signaling and other important cellular processes in single cells that are otherwise challenging to quantitate

Methods

Results

Conclusion