HAP40 targeting macrocyclic peptides: Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) - Part 1 of data set (macrocycle 1)

Differential Hydrogen-Deuterium eXchange Mass Spectrometry (ΔHDX-MS) was performed to determine the binding sites of 8 macrocycles targeting HAP40. To do so, HDX-MS was performed at 0, 0.25, 1, 10 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.本研究通过差示氢氘交换质谱（Differential Hydrogen-Deuterium Exchange Mass Spectrometry，ΔHDX-MS）技术，对8种靶向HAP40的大环化合物的结合位点进行鉴定。为开展本次实验，我们于0、0.25、1、10分钟四个时间点开展HDX-MS检测，所用仪器为搭载HDMSe模式的Waters Select Series Cyclic IMS-MS质谱仪，配套自动化换样液体处理系统（PAL3，Trajan/LEAP）以及搭载HDX技术的ACQUITY UPLC M-Class超高效液相色谱系统（Waters）。氢氘交换标记采用含10 mM磷酸缓冲液（pD 7.5）、150 mM氯化钠的体系；标记完成后，使用含7.5 M盐酸胍、0.5 M三(2-羧乙基)膦（TCEP）、100 mM磷酸缓冲液（pH 2.5）的淬灭液在0℃下终止反应2分钟。淬灭后的样品以1:1比例用100 mM磷酸缓冲液（pH 2.5）稀释后进样，随后采用1:1配比的猪笼草蛋白酶2-胃蛋白酶（Nepenthesin 2-Pepsin，Affipro）进行在线酶解，经脱盐处理后通过反相色谱完成分离。肽段鉴定通过PLGS（ProteinLynx Global Server）完成后，采用DynamX软件分析肽段的氘摄取量。本次提交的质谱原始数据文件格式为Waters .raw格式。