Huntingtin (HTT) targeting macrocyclic peptides: Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) - Part 4 of data set (HHD3)

Differential Hydrogen-Deuterium eXchange Mass Spectrometry (ΔHDX-MS) was performed to determine the binding sites of 5 macrocycles (HL2, HL5, HD4, HHL1, HHD3) targeting Huntingtin (HTT) and HTT-HAP40 complex. To do so, HDX-MS was performed at 0, 0.5, 5, 30 mins using the Waters Select Series Cyclic IMS-MS in HDMSe mode coupled to robotic tool change liquid handling (PAL3, Trajan/LEAP) and ACQUITY UPLC M-Class System with HDX Technology (Waters). HDX labeling was performed using 10 mM Phosphate Buffer pD 7.5, 150 mM NaCl and quenched using 7.5 M Guanidine-HCl, 0.5 M TCEP, 100 mM Phosphate Buffer pH 2.5 at 0 °C for 2 min. These samples were diluted 1:1 with 100 mM Phosphate Buffer, pH 2.5 prior to injection, on-column digestion with 1:1 Nepenthesin 2-Pepsin (Affipro), desalting, and reverse-phase separation. Peptide deuterium uptake was analyzed using DynamX after peptide ID was performed using PLGS. The MS files deposited are in Waters .raw format.本研究采用差示氢氘交换质谱（Differential Hydrogen-Deuterium eXchange Mass Spectrometry，ΔHDX-MS），旨在鉴定针对亨廷顿蛋白（Huntingtin，HTT）及HTT-HAP40复合物的5种大环化合物（HL2、HL5、HD4、HHL1、HHD3）的结合位点。实验中，我们于0、0.5、5、30分钟四个时间点开展氢氘交换质谱（HDX-MS）检测，所用仪器为搭载HDMSe模式的沃特世Select系列循环离子迁移谱质谱（Waters Select Series Cyclic IMS-MS），并配套自动换针液体处理系统（PAL3，Trajan/LEAP）与搭载HDX技术的ACQUITY UPLC M-Class超高效液相色谱系统（ACQUITY UPLC M-Class System with HDX Technology，Waters）。氢氘交换标记采用10 mM磷酸盐缓冲液（pD 7.5）与150 mM氯化钠完成，随后使用7.5 M盐酸胍、0.5 M三(2-羧乙基)膦（TCEP）与100 mM磷酸盐缓冲液（pH 2.5）在0 ℃下淬灭反应2分钟。进样前，样品先用100 mM磷酸盐缓冲液（pH 2.5）以1:1的体积比稀释，随后进行柱内酶解（采用1:1比例的猪笼草蛋白酶2-胃蛋白酶（Nepenthesin 2-Pepsin，Affipro））、脱盐与反相分离。采用PLGS完成肽段鉴定后，使用DynamX软件分析肽段的氘摄入水平。所提交的质谱数据文件均为沃特世（Waters）.raw格式。