Systematic Optimization of the iMALDI Workflow for the Robust and Straightforward Quantification of Signaling Proteins in Cancer Cells.
Proteomics Clin Appl
; 14(5): e2000034, 2020 09.
Article
em En
| MEDLINE
| ID: mdl-32643306
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. EXPERIMENTALDESIGN:
Conditions for tryptic digestion and immuno-enrichment (beads, beadantibody ratios, incubation times, direct vs. indirect immuno-enrichment) are rigorously tested. Different strategies for calibration and data readout are compared.RESULTS:
Digestion using 12 proteintrypsin (wtwt) 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.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Transdução de Sinais
/
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
/
Fluxo de Trabalho
/
Proteínas de Neoplasias
Tipo de estudo:
Guideline
/
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Proteomics Clin Appl
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Canadá