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Immuno-MALDI (iMALDI) for quantifying AKT1 and AKT2 in breast and colorectal cancer cell lines and tumors Robert Popp, Huiyan Li, André LeBlanc, Yassene Mohammed, Adriana Aguilar-Mahecha, Andrew G. Chambers, Cathy Lan, Oliver Poetz, Mark Basik, Gerald Batist, and Christoph H. Borchers Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02934 • Publication Date (Web): 30 Aug 2017 Downloaded from http://pubs.acs.org on August 31, 2017
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Immuno-MALDI (iMALDI) for quantifying AKT1 and AKT2 in breast and colorectal cancer cell lines and tumors Robert Popp1, Huiyan Li1, André LeBlanc2, Yassene Mohammed1,3, Adriana Aguilar-Mahecha4, Andrew G. Chambers1, Cathy Lan5, Oliver Poetz6, Mark Basik5, Gerald Batist5, and Christoph H. Borchers*1,2,5,7 1. University of Victoria - Genome British Columbia Proteomics Centre, University of Victoria, Victoria, British Columbia V8Z 7X8, Canada 2. Proteomics Centre, Segal Cancer 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 Ste-Catherine Road, Montreal, Quebec, H3T 1E2, Canada 5. Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, 5100 de Maisonneuve Blvd. West, Suite 720, Montreal, Quebec, H4A 3T2, Canada 6. Natural and Medical Sciences Institute (NMI) at the University of Tübingen, Markwiesenstr, 55, Reutlingen 72074, Germany 7. Department of Biochemistry and Microbiology, University of Victoria, Petch Building, Room 270d, 3800 Finnerty Rd., Victoria, British Columbia, V8Z 7X8, Canada.
*Corresponding Author: Dr. Christoph H. Borchers University of Victoria – Genome BC Proteomics Centre 3101-4464 Markham St, Victoria, BC V8Z 7X8, Canada Telephone +1 250 483-3221; Fax +1 250 483-3238 Email:
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Keywords: iMALDI; immuno-MALDI; breast cancer; colorectal cancer; protein quantitation; patient stratification; AKT1; AKT2 Running title: immuno-MALDI (iMALDI) assay for AKT1 and AKT2
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List of Abbreviations AAA, Amino acid analysis ACN, Acetonitrile AAA, Amino acid analysis ACN, Acetonitrile AKT, Protein kinase B BCA, Bicinchoninic acid CZE, Capillary zone electrophoresis END, Endogenous peptide FA, Formic acid FBS, Fetal bovine serum FDA, U.S. Food and Drug Administration IHC, Immunohistochemistry iMALDI, immuno-matrix assisted laser desorption/ionization JGH, Jewish General Hospital LC-MS, Liquid chromatography mass spectrometry MRM, Multiple reaction monitoring MS, Mass spectrometry mTOR, Mechanistic target of rapamycin NAT, Natural (light) version of a peptide NMI, Natural and Medical Sciences Institute PI3K, Phosphatidylinositol-3-kinase PTEN, Phosphatase and tensin homolog PTM, Post-translational modification RPMI, Roswell Park Memorial Institute ACS Paragon Plus Environment
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SIS, Stable isotope-labeled standard TOF, Time of flight
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Abstract The PI3K/AKT/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 (LC/ESI-MRM) are more commonly used in clinical research. Both technologies have certain disadvantages, namely the non-specificity 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 to 2.0 fmol/µg total lysate protein, and with CVs
