Differential Proteomic Shotgun Analysis Elucidates Involvement of Water Channel Aquaporin 8 in Presence of α-Amylase in the Colon

Aquaporin (AQP) family plays a pivotal role in fluid secretion and absorption, especially in the digestive system and secretory glands. Within this family, AQP8 was reported to be widely expressed in the epithelia of the digestive tract, liver, and pancreas. In two parallel experimental platforms with different analytical and comparative approaches, in-gel tryptic digestion with macro-embedded spreadsheet analysis and in-solution tryptic digestion with LC−MS alignment based approach, we compared wild-type and AQP8 knockout mice colon proteomes. Shared result between both experiments revealed down-regulation of α-amylase 2 in AQP8-deleted mice model. Verification on both transcriptional and translational levels confirmed the involvement of AQP8 in α-amylase 2 regulation. Given the profound role of AQP8 as a water and solutes transporter, it might be important in modulating α-amylase 2 synthesis by colonic epithelial cells as well. Here, we also proved the capability of our coupled approaches for selecting the most reliable and significant candidates, an applicable process for initial screening of biological biomarkers in complex specimens and tissue extracts.

水通道蛋白（Aquaporin，AQP）家族在体液分泌与吸收过程中发挥关键作用，尤其在消化系统与分泌腺中。该家族成员水通道蛋白8（AQP8）被报道广泛表达于消化道、肝脏及胰腺的上皮组织内。本研究采用两个分析与比较策略各异的平行实验平台：其一为凝胶内胰蛋白酶消化结合宏嵌入电子表格分析法，其二为溶液内胰蛋白酶消化结合基于液相色谱-质谱联用法（LC-MS）的比对分析法，对比了野生型与AQP8基因敲除小鼠的结肠蛋白质组。两项实验的共同结果显示，AQP8缺失小鼠模型中α-淀粉酶2（α-amylase 2）的表达量显著下调。通过转录与翻译水平的验证实验，证实了AQP8参与调控α-淀粉酶2的表达。鉴于AQP8作为水与溶质转运蛋白的核心功能，其可能同样在结肠上皮细胞的α-淀粉酶2合成调控中发挥重要作用。本研究同时证实，所采用的联用方法可筛选出最具可靠性与显著性的候选分子，该流程可应用于复杂标本与组织提取物中生物标志物（biological biomarkers）的初步筛选。