Immuno-Matrix-Assisted Laser Desorption/Ionization Assays for Quantifying AKT1 and AKT2 in Breast and Colorectal Cancer Cell Lines and Tumors
| Autor: | Oliver Poetz, Adriana Aguilar-Mahecha, Yassene Mohammed, Mark Basik, Gerald Batist, Robert Popp, Cathy Lan, Andrew G. Chambers, Christoph H. Borchers, André LeBlanc, Huiyan Li |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Electrospray ionization Quantitative proteomics Breast Neoplasms Mass spectrometry Analytical Chemistry 03 medical and health sciences Limit of Detection Cell Line Tumor Humans Amino Acid Sequence Protein kinase B Mechanistic target of rapamycin Chromatography High Pressure Liquid PI3K/AKT/mTOR pathway biology Chemistry Selected reaction monitoring Molecular biology 3. Good health Matrix-assisted laser desorption/ionization 030104 developmental biology Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein Cancer research Female Colorectal Neoplasms Peptides Antibodies Immobilized Proto-Oncogene Proteins c-akt |
| Zdroj: | Analytical Chemistry, 89(19), 10592-10600 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/acs.analchem.7b02934 |
| Popis: | 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 variation15%. 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. |
| Databáze: | OpenAIRE |
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