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Anal Chem. 2017 Oct 3;89(19):10592-10600. doi: 10.1021/acs.analchem.7b02934. Epub 2017 Sep 20.

Immuno-Matrix-Assisted Laser Desorption/Ionization Assays for Quantifying AKT1 and AKT2 in Breast and Colorectal Cancer Cell Lines and Tumors.

Author information

1
University of Victoria Genome British Columbia Proteomics Centre, University of Victoria , Victoria, British Columbia V8Z 7X8, Canada.
2
Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University , 3755 Côte-Sainte-Catherine Road, Montreal, Quebec H3T 1E2, Canada.
3
Center for Proteomics and Metabolomics, Leiden University Medical Center , Albinusdreef 2, Leiden, 2333 ZA, The Netherlands.
4
Lady Davis Institute, Jewish General Hospital, McGill University , 3755 Côte-Sainte-Catherine Road, Montreal, Quebec H3T 1E2, Canada.
5
Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University , 5100 de Maisonneuve Boulevard West, Suite 720, Montreal, Quebec H4A 3T2, Canada.
6
Natural and Medical Sciences Institute, University of Tübingen , Markwiesenstrasse 55, Reutlingen 72074, Germany.
7
Department of Biochemistry and Microbiology, University of Victoria , Petch Building, Room 270d, 3800 Finnerty Road, Victoria, British Columbia, V8P 5C2, Canada.

Abstract

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway is one of the most commonly dysregulated signaling pathways that is linked to cancer development and progression, and its quantitative protein analysis holds the promise to facilitate patient stratification for targeted therapies. Whereas immunohistochemistry (IHC) and immunoassays are routinely used for clinical analysis of signaling pathways, mass spectrometry-based approaches such as liquid chromatography/electrospray ionization multiple reaction monitoring mass spectrometry (LC/ESI-MRM-MS) are more commonly used in clinical research. Both technologies have certain disadvantages, namely, the nonspecificity of IHC and immunoassays, and potentially long analysis times per sample of LC/ESI-MRM-MS. To create a robust, fast, and sensitive protein quantification tool, we developed immuno-matrix-assisted laser desorption/ionization (iMALDI) assays with automated liquid handling. The assays are able to quantify AKT1 and AKT2 from breast cancer and colon cancer cell lines and flash-frozen tumor lysates with a linear range of 0.05-2.0 fmol/μg of total lysate protein and with coefficients of variation < 15%. Compared to other mass spectrometric methods, the developed assays require less sample per analysis-only 25 μg of total protein-and are therefore suitable for analysis of needle biopsies. Furthermore, the presented iMALDI technique is the first MS-based method for absolute quantitation of AKT peptides from cancer tissues. This study demonstrates the suitability of iMALDI for low limit-of-detection and reproducible quantitation of signaling pathway members using a benchtop MALDI mass spectrometer within approximately 6-7 h.

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