A multiplexed, automated immuno-matrix assisted laser desorption/ionization mass spectrometry assay for simultaneous and precise quantitation of PTEN and p110α in cell lines and tumor tissues

The Analyst, vol. 146, pp. 6566–6575

Abstract

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN is often down-regulated in cancer, and there are conflicting data on PTEN's role as breast cancer biomarker. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody crossreactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. Our iMALDI assay only requires a low-cost benchtop MALDI-TOF mass spectrometer, which simplifies clinical translation. We validated our assay's precision and accuracy, with simultaneous enrichment of both target proteins not significantly affecting the precision and accuracy of the quantitation when compared to the PTEN- and p110α-singleplex iMALDI assays (<15% difference). The multiplexed assay's linear range is from 0.6-20 fmol with accuracies of 90-112% for both target proteins, and the assay is free of matrix-related interferences. The inter-day reproducibility over 5-days was high, with an overall CV of 9%. PTEN and p110α protein concentrations can be quantified down to 1.4 fmol and 0.6 fmol per 10 μg of total tumor protein, respectively, in various tumor tissue samples, including fresh-frozen breast tumors and colorectal cancer liver metastases, and patient-derived xenograft (PDX) tumors.

Authors 14

1. Bjoern C. Froehlich

   University of Victoria

   Affiliation as printed

   Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 5C2, Canada

   University of Victoria – Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia V8Z 7X8, Canada

2. Robert Popp

   University of Victoria

   Affiliation as printed

   University of Victoria – Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia V8Z 7X8, Canada

3. Constance A. Sobsey

   Jewish General Hospital · Lady Davis Institute for Medical Research · McGill University

   Affiliation as printed

   Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T1E2, Canada

4. Andre M. LeBlanc

   Jewish General Hospital · Lady Davis Institute for Medical Research · McGill University

   Affiliation as printed

   Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T1E2, Canada

5. Yassene Mohammed Aachen Center for Proteomics and Metabolomics

   Skolkovo Institute of Science and Technology · University of Victoria · Leiden University Medical Center

   Affiliation as printed

   Center for Computational and Data-Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Moscow 121205, Russia

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

   University of Victoria – Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia V8Z 7X8, Canada

6. Marguerite Buchanan

   Jewish General Hospital · Lady Davis Institute for Medical Research · McGill University

   Affiliation as printed

   Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T1E2, Canada

7. Adriana Aguilar‐Mahecha

   Jewish General Hospital · Lady Davis Institute for Medical Research · McGill University

   Affiliation as printed

   Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T1E2, Canada

8. Oliver Poetz

   Natural and Medical Sciences Institute · Signatope GmbH (Germany) · University of Tübingen

   Affiliation as printed

   NMI Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen 72770, Germany

   SIGNATOPE GmbH, Reutlingen 72770, Germany

9. Michael X. Chen

   University of British Columbia

   Affiliation as printed

   Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, Vancouver, Canada

10. Alan Spatz

    Jewish General Hospital · Lady Davis Institute for Medical Research · McGill University

    Affiliation as printed

    Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T1E2, Canada

11. Mark Basik

    Jewish General Hospital · Lady Davis Institute for Medical Research · McGill University

    Affiliation as printed

    Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T1E2, Canada

12. Gerald Batist

    Jewish General Hospital · Lady Davis Institute for Medical Research · McGill University

    Affiliation as printed

    Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC H4A3T2, Canada

    Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T1E2, Canada

13. René Peiman Zahedi

    Skolkovo Institute of Science and Technology · Jewish General Hospital · Lady Davis Institute for Medical Research · McGill University

    Affiliation as printed

    Center for Computational and Data-Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Moscow 121205, Russia

    Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T1E2, Canada

14. Christoph H. Borchers corresponding

    Skolkovo Institute of Science and Technology · University of Victoria · Jewish General Hospital · Lady Davis Institute for Medical Research · McGill University

    Affiliation as printed

    Center for Computational and Data-Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Moscow 121205, Russia

    Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 5C2, Canada

    Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC H4A3T2, Canada

    Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T1E2, Canada

    University of Victoria – Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia V8Z 7X8, Canada