Systematic Optimization of the iMALDI Workflow for the Robust and Straightforward Quantification of Signaling Proteins in Cancer Cells
Open accessAbstract
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.
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- Authors
- Froehlich, B. C., Popp, R., Sobsey, C. A., Ibrahim, S., LeBlanc, A. M., Mohammed, Y., Aguilar‐Mahecha, A., Poetz, O., Chen, M. X., Spatz, A., Basik, M., Batist, G., Zahedi, R. P., & Borchers, C. H.
- Affiliations
- University of Victoria (CA)
- Jewish General Hospital (CA)
- Skolkovo Institute of Science and Technology (RU)
- Natural and Medical Sciences Institute (DE)
- University of British Columbia (CA)
- Corresponding author
- Borchers, Christoph H.
- ORCID
- Venue
- PROTEOMICS – Clinical Applications
- Publisher
- Wiley
- Published
- 2020-08-09
- Volume
- 14
- Issue
- 5
- Pages
- e2000034-e2000034
- ISSN
- 1862-8346 (ISSN-L)
- 1862-8354
- PMC
- PMC7539945
- OpenAlex
- W3042184853
- Cited by
- 9
- References
- 35
- Citations by year
- 2025: 1 · 2024: 2 · 2023: 2 · 2022: 1 · 2021: 1 · 2020: 2
- FWCI
- 0.57
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- PROTEOMICS - CLINICAL APPLICATIONS (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Data Archiving and Networked Services (DANS) (submittedVersion, cc-by-nc-nd)
- Leiden Repository (Leiden University) (submittedVersion, cc-by-nc-nd)
- Funders
- Leading Edge Endowment Fund
- Genome British Columbia (183AKT, 204PRO, 214PRO, 264PRO)
- Genome Canada (183AKT, 204PRO, 214PRO, 264PRO, PD‐L1)
- Terry Fox Research Institute
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced Biosensing Techniques and Applications
- Keywords
- protein quantification, PTEN, trypsin digestion, peptide recovery, calibration matrix, assay automation, cancer cells
- MeSH terms
- Humans, Neoplasm Proteins, Time Factors, Signal Transduction, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Cell Line, Tumor, Workflow, Limit of Detection
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