Identification and quantification of AKT isoforms and phosphoforms in breast cancer using a novel nanofluidic immunoassay

Demetris C. Iacovides, Aimee B. Johnson, Nick Wang, Shanta Boddapati, James Korkola, Joe Gray

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Breast cancer subtype-specific molecular variations can dramatically affect patient responses to existing therapies. It is thought that differentially phosphorylated protein isoforms might be a useful prognostic biomarker of drug response in the clinic. However, the accurate detection and quantitative analysis of cancer-related protein isoforms and phospho-isoforms in tumors are limited by current technologies. Using a novel, fully automated nanocapillary electrophoresis immunoassay (NanoProTM 1000) designed to separate protein molecules based on their isoelectric point, we developed a reliable and highly sensitive assay for the detection and quantitation of AKT isoforms and phosphoforms in breast cancer. This assay enabled the measurement of activated AKT1/2/3 in breast cancer cells using protein produced from as few as 56 cells. Importantly, we were able to assign an identity for the phosphorylated S473 phosphoform of AKT1, the major form of activated AKT involved in multiple cancers, including breast, and a current focus in clinical trials for targeted intervention. The ability of our AKT assay to detect and measure AKT phosphorylation from very low amounts of total protein will allow the accurate evaluation of patient response to drugs targeting activated PI3K-AKT using scarce clinical specimens. Moreover, the capacity of this assay to detect and measure all three AKT isoforms using one single pan-specific antibody enables the study of the multiple and variable roles that these isoforms play in AKT tumorigenesis.

Original languageEnglish (US)
Pages (from-to)3210-3220
Number of pages11
JournalMolecular and Cellular Proteomics
Volume12
Issue number11
DOIs
StatePublished - Nov 2013

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Nanofluidics
Immunoassay
Protein Isoforms
Breast Neoplasms
Assays
Pharmacological Biomarkers
Phosphorylation
Proteins
Isoelectric Point
Drug Delivery Systems
Electrophoresis
Phosphatidylinositol 3-Kinases
Tumors
Neoplasms
Carcinogenesis
Cells
Clinical Trials
Technology
Molecules
Antibodies

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Cite this

Identification and quantification of AKT isoforms and phosphoforms in breast cancer using a novel nanofluidic immunoassay. / Iacovides, Demetris C.; Johnson, Aimee B.; Wang, Nick; Boddapati, Shanta; Korkola, James; Gray, Joe.

In: Molecular and Cellular Proteomics, Vol. 12, No. 11, 11.2013, p. 3210-3220.

Research output: Contribution to journalArticle

Iacovides, Demetris C. ; Johnson, Aimee B. ; Wang, Nick ; Boddapati, Shanta ; Korkola, James ; Gray, Joe. / Identification and quantification of AKT isoforms and phosphoforms in breast cancer using a novel nanofluidic immunoassay. In: Molecular and Cellular Proteomics. 2013 ; Vol. 12, No. 11. pp. 3210-3220.
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