On-Tissue Chemical Oxidation Followed by Derivatization for Mass Spectrometry Imaging Enables Visualization of Primary and Secondary Hydroxyl-Containing Metabolites in Biological Tissues

On-tissue chemical derivatization combined with mass spectrometry imaging (MSI) can effectively visualize low-abundance and poorly ionizable molecules in biological tissues. Owing to the lack of an effective chemical reaction environment on the tissue surface, the development of direct one-step derivatization reactions is challenging. Herein, we present a two-step reaction involving on-tissue chemical oxidation followed by derivatization combined with airflow-assisted desorption electrospray ionization-MSI, enabling the visualization of primary and secondary hydroxyl-containing metabolites (PSHMs) within the tissue sections. This method indirectly achieved on-tissue derivatization by combining two reactions. Hydroxyl was converted to carbonyl using chemical oxidants, and subsequently, carbonyl was derived using Girard’s P reagent. Using this methodology, 169 PSHMs, including hydroxy fatty acids (OH–FAs), fatty alcohols (FOHs), and sterol lipids, were detected and imaged in the tissues of rat brain, kidney, and liver. Moreover, we found that the abundant PSHMs, fatty aldehydes, and oxo fatty acids were significantly dysregulated in the liver and kidney tissues of type 2 diabetic rats; in particular, OH–FAs and FOHs were remarkably up-regulated in the diabetic rat liver tissues. The aberrations of these oxidative metabolites provide insights into the understanding of the molecular pathological mechanism of diabetes. This study demonstrates a novel, two-step reaction strategy for on-tissue derivatization with the analysis of previously inaccessible molecules using MSI.组织原位化学衍生化结合质谱成像（MSI）可有效可视化生物组织中的低丰度、难电离分子。由于组织表面缺乏有效的化学反应环境，开发直接一步法衍生反应颇具挑战。本研究提出一种两步反应策略：先开展组织原位化学氧化，再结合气流辅助解吸电喷雾电离-质谱成像（airflow-assisted desorption electrospray ionization-MSI）完成衍生化，从而实现组织切片中伯仲羟基类代谢物（PSHMs）的可视化检测。该方法通过两步反应间接实现组织原位衍生化：先利用化学氧化剂将羟基转化为羰基，随后通过吉拉德P试剂（Girard’s P reagent）对羰基进行衍生标记。采用该方法，我们在大鼠脑、肾及肝组织中成功检测并成像得到169种伯仲羟基类代谢物，涵盖羟基脂肪酸（OH–FAs）、脂肪醇（FOHs）与甾醇脂质。此外，我们观察到2型糖尿病大鼠肝、肾组织中丰度较高的伯仲羟基类代谢物、脂肪醛及氧代脂肪酸均存在显著失调；尤为关键的是，糖尿病大鼠肝组织中的羟基脂肪酸与脂肪醇呈现显著上调趋势。这些氧化代谢物的异常变化为解析糖尿病的分子病理机制提供了重要研究视角。本研究提出了一种新颖的两步法组织原位衍生化策略，可实现此前难以通过质谱成像分析的分子的检测与成像。