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

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.

Precise Quantitation of PTEN by Immuno-MRM: A Tool To Resolve the Breast Cancer Biomarker Controversy

“…Immuno-MRM Boosts the Sensitivity of PTEN Quantitation and Shows High Accuracy. We decided to generate an antipeptide antibody targeted against our proteotypic PTEN peptide NNIDDVVR in order to develop an immuno-mass spectrometry assay with superior sensitivity, as we have previously done to quantify other cancer signaling proteins, such as AKT1, AKT2, 48 and PI3K, 49 by immuno-MALDI mass spectrometry.…”

Prospective on Imaging Mass Spectrometry in Clinical Diagnostics

“…Enriched peptides can be eluted directly onto the MALDI target, minimizing sample loss due to handling tasks ( 15 ). Assays using iMALDI have detected glycosylated transferrin, a marker of alcoholism, from human serum ( 17 ) and screened human plasma for primary aldosteronism, which is clinically associated with hypertension ( 15 , 18 , 19 ). These assays have the potential to be multiplexed, further strengthening the applicability of these assays to clinical laboratories ( 15 , 20 , 21 ).…”

Using Two Peptide Isotopologues as Internal Standards for the Streamlined Quantification of Low-Abundance Proteins by Immuno-MRM and Immuno-MALDI

“…Even when external calibration curves were generated from the same sample type after immuno-enrichment, endogenous signals may still interfere . Using surrogate matrices to generate external calibration curves, such as bovine serum albumin (BSA) or Escherichia coli digests, has previously been shown to be feasible but may not sufficiently represent interference and suppression events occurring in clinical samples, including FFPE specimens, where considerable unanticipated off-target binding of antibodies may occur. Proving that a particular external calibration works for a given combination of immuno-MS assay and patient tissue sample on a case-by-case basis is virtually impossible.…”