Systematic Optimization of the iMALDI Workflow for the Robust and Straightforward Quantification of Signaling Proteins in Cancer Cells
PROTEOMICS - CLINICAL APPLICATIONS, vol. 14, pp. e2000034
Abstract
PURPOSE: Immuno-MALDI (iMALDI) combines immuno-enrichment of biomarkers with MALDI-MS for fast, precise, and specific quantitation, making it a valuable tool for developing clinical assays. iMALDI assays are optimized for the PI3-kinase signaling pathway members phosphatase and tensin homolog (PTEN) and PI3-kinase catalytic subunit alpha (p110α), with regard to sensitivity, robustness, and throughput. A standardized template for developing future iMALDI assays, including automation protocols to streamline assay development and translation, is provided. EXPERIMENTAL DESIGN: Conditions for tryptic digestion and immuno-enrichment (beads, bead:antibody ratios, incubation times, direct vs. indirect immuno-enrichment) are rigorously tested. Different strategies for calibration and data readout are compared. RESULTS: Digestion using 1:2 protein:trypsin (wt:wt) for 1 h yielded high and consistent peptide recoveries. Direct immuno-enrichment (antibody-bead coupling prior to antigen-enrichment) yielded 30% higher peptide recovery with a 1 h shorter incubation time than indirect enrichment. Immuno-enrichment incubation overnight yielded 1.5-fold higher sensitivities than 1 h incubation. Quantitation of the endogenous target proteins is not affected by the complexity of the calibration matrix, further simplifying the workflow. CONCLUSIONS AND CLINICAL RELEVANCE: This optimized and automated workflow will facilitate the clinical translation of high-throughput sensitive iMALDI assays for quantifying cell-signaling proteins in individual tumor samples, thereby improving patient stratification for targeted treatment.
Authors 13
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Affiliation as printed
Department of Biochemistry and Microbiology University of Victoria Victoria BC V8P 5C2 Canada
University of Victoria‐Genome BC Proteomics Centre University of Victoria Victoria BC V8Z 7E8 Canada
Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8P 5C2 Canada
University of Victoria-Genome BC Proteomics Centre, University of Victoria, Victoria, BC, V8Z 7E8 Canada
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Affiliation as printed
University of Victoria‐Genome BC Proteomics Centre University of Victoria Victoria BC V8Z 7E8 Canada
University of Victoria-Genome BC Proteomics Centre, University of Victoria, Victoria, BC, V8Z 7E8 Canada
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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
Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, H3T1E2 Canada
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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
Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, H3T1E2 Canada
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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 2333 ZA The Netherlands
University of Victoria‐Genome BC Proteomics Centre University of Victoria Victoria BC V8Z 7E8 Canada
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, 2333 ZA The Netherlands
University of Victoria-Genome BC Proteomics Centre, University of Victoria, Victoria, BC, V8Z 7E8 Canada
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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
Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, H3T1E2 Canada
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Natural and Medical Sciences Institute · Signatope GmbH (Germany) · University of Tübingen
Affiliation as printed
NMI Natural and Medical Sciences Institute University of Tuebingen Reutlingen 72770 Germany
SIGNATOPE GmbH Reutlingen 72770 Germany
NMI Natural and Medical Sciences Institute University of Tuebingen, Reutlingen, 72770 Germany
SIGNATOPE GmbH, Reutlingen, 72770 Germany
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University of British Columbia
Affiliation as printed
Department of Pathology and Laboratory Medicine Faculty of Medicine University of British Columbia Vancouver Canada
Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, Vancouver, Canada
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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
Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, H3T1E2 Canada
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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
Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, H3T1E2 Canada
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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
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
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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
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
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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
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 BC V8Z 7E8 Canada
Center for Computational and Data-Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Moscow, 121205 Russia
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, BC, V8Z 7E8 Canada
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