Dataset Information

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

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.

SUBMITTER: Jacob T

PROVIDER: S-EPMC4913309 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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

Jacob Thomas T Agarwal Anupriya A Ramunno-Johnson Damien D O'Hare Thomas T Gönen Mehmet M Tyner Jeffrey W JW Druker Brian J BJ Vu Tania Q TQ

Scientific reports 20160620

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. ...[more]

PMID: 27320899

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Project description:Phenology-the timing of life-history events-is a key trait for understanding responses of organisms to climate. The digitization and online mobilization of herbarium specimens is rapidly advancing our understanding of plant phenological response to climate and climatic change. The current practice of manually harvesting data from individual specimens, however, greatly restricts our ability to scale-up data collection. Recent investigations have demonstrated that machine-learning approaches can facilitate this effort. However, present attempts have focused largely on simplistic binary coding of reproductive phenology (e.g., presence/absence of flowers). Here, we use crowd-sourced phenological data of buds, flowers, and fruits from >3,000 specimens of six common wildflower species of the eastern United States (Anemone canadensis L., A. hepatica L., A. quinquefolia L., Trillium erectum L., T. grandiflorum (Michx.) Salisb., and T. undulatum Wild.) to train models using Mask R-CNN to segment and count phenological features. A single global model was able to automate the binary coding of each of the three reproductive stages with >87% accuracy. We also successfully estimated the relative abundance of each reproductive structure on a specimen with ≥90% accuracy. Precise counting of features was also successful, but accuracy varied with phenological stage and taxon. Specifically, counting flowers was significantly less accurate than buds or fruits likely due to their morphological variability on pressed specimens. Moreover, our Mask R-CNN model provided more reliable data than non-expert crowd-sourcers but not botanical experts, highlighting the importance of high-quality human training data. Finally, we also demonstrated the transferability of our model to automated phenophase detection and counting of the three Trillium species, which have large and conspicuously-shaped reproductive organs. These results highlight the promise of our two-phase crowd-sourcing and machine-learning pipeline to segment and count reproductive features of herbarium specimens, thus providing high-quality data with which to investigate plant responses to ongoing climatic change.

Dataset Information

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

Publications

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

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