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.
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.