Immuno-MALDI(iMALDI)将生物标志物的免疫富集与MALDI-MS相结合,精确,和特定的定量,使其成为开发临床试验的有价值的工具。iMALDI分析针对PI3激酶信号通路成员磷酸酶和张力蛋白同源物(PTEN)和PI3激酶催化亚基α(p110α)进行了优化,关于敏感性,鲁棒性,和吞吐量。用于开发未来iMALDI测定的标准化模板,包括自动化协议,以简化分析开发和翻译,提供。
胰蛋白酶消化和免疫富集的条件(珠子,珠子:抗体比率,孵化时间,直接vs.间接免疫富集)经过严格测试。比较了校准和数据读出的不同策略。
使用1:2蛋白质:胰蛋白酶(wt:wt)消化1小时产生高且一致的肽回收率。与间接富集相比,直接免疫富集(抗原富集之前的抗体-珠子偶联)产生了30%的肽回收率,孵育时间短1小时。免疫富集孵育过夜产生的敏感性比孵育1小时高1.5倍。内源性靶蛋白的定量不受校准矩阵的复杂性的影响。进一步简化工作流程。
这种优化和自动化的工作流程将促进高通量敏感iMALDI测定的临床翻译,用于定量单个肿瘤样品中的细胞信号蛋白。从而改善患者分层进行针对性治疗。
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.
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.
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.
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.