Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115, United States.
Anal Chem. 2022 Jul 19;94(28):10142-10150. doi: 10.1021/acs.analchem.2c01446. Epub 2022 Jul 7.
During the analysis steps of hydrogen-deuterium exchange (HDX) mass spectrometry (MS), there is an unavoidable loss of deuterons, or back-exchange. Understanding back-exchange is necessary to correct for loss during analysis, to calculate the absolute amount of exchange, and to ensure that deuterium recovery is as high as possible during liquid chromatography (LC)-MS. Back-exchange can be measured and corrected for using a maximally deuterated species (here called maxD), in which the protein is deuterated at positions and analyzed with the same buffer components, %DO, quenching conditions, and LC-MS parameters used during the analysis of other labeled samples. Here, we describe a robust and broadly applicable protocol, using denaturation followed by deuteration, to prepare a maxD control sample in ∼40 min for nonmembrane proteins. The protocol was evaluated with a number of proteins that varied in both size and folded structure. The relative fractional uptake and level of back-exchange with this protocol were both equivalent to those obtained with earlier protocols that either require much more time or require isolation of peptic peptides prior to deuteration. Placing strong denaturation first in the protocol allowed for maximum deuteration in a short time (∼10 min) with equal or more deuteration found in other methods. The absence of high temperatures and low pH during the deuteration step limited protein aggregation. This high-performance, fast, and easy-to-perform protocol should enhance routine preparation of maxD controls.
在氢氘交换（HDX）质谱（MS）的分析步骤中，不可避免地会损失氘原子，即回交换。了解回交换对于在分析过程中进行损失校正、计算绝对交换量以及确保在液相色谱（LC）-MS 过程中尽可能高地回收氘是必要的。可以使用最大限度氘化的物质（这里称为 maxD）来测量和校正回交换，其中蛋白质在位置上被氘化，并使用与分析其他标记样品时相同的缓冲成分、%DO、淬灭条件和 LC-MS 参数进行分析。在这里，我们描述了一种稳健且广泛适用的方案，使用变性 followed by 氘化，在大约 40 分钟内为非膜蛋白制备 maxD 对照样品。该方案用许多大小和折叠结构不同的蛋白质进行了评估。与需要更多时间或需要在氘化之前分离肽酶肽的早期方案相比，该方案的相对分数摄取和回交换水平都等效。在方案中首先进行强烈变性允许在短时间内（约 10 分钟）进行最大氘化，并且在其他方法中发现的氘化程度相等或更高。在氘化步骤中没有高温和低 pH 限制了蛋白质聚集。这种高性能、快速且易于执行的方案应增强 maxD 对照品的常规制备。
