In this work, the sorbent-attached microfunnels used in funnel-based spray ionization mass spectrometry were evaluated for the all-in-one digestion of proteins. Sorbent materials, including C18 and TiO2 powders, were used as substrates to support in-funnel digestion and subsequent solid-phase extraction and purification of the digested products. In-funnel digestion protocols with and without reductive alkylation were developed for the analysis of proteins with and without disulfide linkages. Compared with in-solution digestion of the same loadings, the sequence coverage of in-funnel digestion of ovalbumin (with one disulfide bond) and ovocystatin (with two disulfide bonds) increased from 36% to 65% and from 21% to 81%, respectively. Loading 100 fmol of ovalbumin was sufficient to generate detectable tryptic fragments on C18-attached funnels. Notably, some phosphorylated digestion fragments were solely detected on C18-attached funnels and some nonphosphorylated digestion fragments were detected only on TiO2-attached funnels. Complex biological protein mixtures (i.e., bovine milk) and mouse liver protein extract could also be digested on C18- and TiO2-attached funnels. Using this platform, 30 samples were digested at the same time with enhanced digestion efficiency and were analyzed by funnel-based spray ionization mass spectrometry. This approach is potentially useful for sensitive and high-throughput bottom-up proteomic studies of complex biological samples.

中文翻译：

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使用基于吸附剂膜漏斗的喷雾电离质谱法开发多合一蛋白质消化平台。

在这项工作中，评估了用于基于漏斗的喷雾电离质谱法的吸附剂连接微漏斗对蛋白质的多合一消化。吸附剂材料，包括 C18 和 TiO2 粉末，被用作支持漏斗内消化和随后消化产物的固相萃取和纯化的底物。开发了带有和不带有还原烷基化的漏斗内消化方案，用于分析带有和不带有二硫键的蛋白质。与相同负载的溶液内消化相比，卵清蛋白（具有一个二硫键）和卵胱抑素（具有两个二硫键）的漏斗内消化的序列覆盖率从 36% 增加到 65% 和从 21% 增加到 81%，分别。加载 100 fmol 的卵清蛋白足以在 C18 连接的漏斗上产生可检测的胰蛋白酶片段。值得注意的是，一些磷酸化消化片段仅在连接 C18 的漏斗上检测到，而一些非磷酸化消化片段仅在连接 TiO2 的漏斗上检测到。复杂的生物蛋白质混合物（即牛乳）和小鼠肝脏蛋白质提取物也可以在 C18 和 TiO2 连接的漏斗上消化。使用该平台，30 个样品同时消解，消解效率更高，并通过基于漏斗的喷雾电离质谱法进行分析。这种方法对于复杂生物样品的敏感和高通量自下而上的蛋白质组学研究可能有用。牛乳）和小鼠肝脏蛋白质提取物也可以在 C18 和 TiO2 连接的漏斗上消化。使用该平台，30 个样品同时消解，消解效率更高，并通过基于漏斗的喷雾电离质谱法进行分析。这种方法对于复杂生物样品的敏感和高通量自下而上的蛋白质组学研究可能有用。牛乳）和小鼠肝脏蛋白质提取物也可以在 C18 和 TiO2 连接的漏斗上消化。使用该平台，30 个样品同时消解，消解效率更高，并通过基于漏斗的喷雾电离质谱法进行分析。这种方法对于复杂生物样品的敏感和高通量自下而上的蛋白质组学研究可能有用。