Systematic Optimization of the iMALDI Workflow for the Robust and Straightforward Quantification of Signaling Proteins in Cancer Cells

Proteomics Clin Appl. 2020 Sep;14(5):e2000034. doi: 10.1002/prca.202000034. Epub 2020 Aug 9.


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.

Keywords: clinical mass spectrometry; formalin-fixed paraffin-embedded (FFPE); immunohistochemistry; protein assays; translation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Humans
  • Limit of Detection
  • Neoplasm Proteins / metabolism*
  • Signal Transduction*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*
  • Time Factors
  • Workflow*


  • Neoplasm Proteins