Systematic Optimization of the iMALDI Workflow for the Robust and Straightforward Quantification of Signaling Proteins in Cancer Cells.
Systematic Optimization of the iMALDI Workflow for the Robust and Straightforward Quantification of Signaling Proteins in Cancer Cells.
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DOI：
10.1002/prca.202000034
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发表时间：
2020-09
期刊：
影响因子：
--
通讯作者：
Borchers CH
中科院分区：
文献类型：
--
作者：
Froehlich BC;Popp R;Sobsey CA;Ibrahim S;LeBlanc AM;Mohammed Y;Aguilar-Mahecha A;Poetz O;Chen MX;Spatz A;Basik M;Batist G;Zahedi RP;Borchers CH
免疫 MALDI (iMALDI) 将生物标志物的免疫富集与 MALDI-MS 相结合，可实现快速、精确和特异的定量，使其成为开发临床检测的宝贵工具。 iMALDI 检测针对 PI3 激酶信号通路成员磷酸酶和张力蛋白同源物 (PTEN) 以及 PI3 激酶催化亚基 α (p110α) 在灵敏度、稳健性和通量方面进行了优化。提供了用于开发未来 iMALDI 检测的标准化模板，包括简化检测开发和翻译的自动化协议。胰蛋白酶消化和免疫富集的条件（珠子、珠子：抗体比率、孵育时间、直接与间接免疫富集）经过严格测试。比较了不同的校准和数据读出策略。使用 1:2 蛋白质:胰蛋白酶 (wt:wt) 消化 1 小时，获得高且一致的肽回收率。与间接富集相比，直接免疫富集（抗原富集之前抗体珠偶联）的肽回收率提高了 30%，孵育时间缩短了 1 小时。免疫富集孵育过夜的灵敏度比孵育 1 小时高 1.5 倍。内源靶蛋白的定量不受校准矩阵复杂性的影响，进一步简化了工作流程。这种优化的自动化工作流程将促进高通量敏感 iMALDI 检测的临床转化，用于量化单个肿瘤样本中的细胞信号蛋白，从而改善靶向治疗的患者分层。
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.
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