Neuropeptides are an important class of endogenous peptides in the nervous system that regulate physiological functions such as feeding, glucose homeostasis, pain, memory, reproduction, and many others. In order to understand the functional role of neuropeptides in diseases or disorders, studies investigating their dysregulation in terms of changes in abundance and localization must be carried out. As multiple neuropeptides are believed to play a functional role in each physiological process, techniques capable of global profiling multiple neuropeptides simultaneously are desired. Mass spectrometry is well-suited for this goal due to its ability to perform untargeted measurements without prior comprehensive knowledge of the analytes of interest. Mass spectrometry imaging (MSI) is particularly useful because it has the capability to image a large variety of peptides in a single experiment without labeling. Like all analytical techniques, careful sample preparation is critical to successful MSI analysis. The first half of this review focuses on recent developments in MSI sample preparation and instrumentation for analyzing neuropeptides and other biomolecules in which the sample preparation technique may be directly applicable for neuropeptide analysis. The benefit offered by incorporating these techniques is shown as improvement in a number of observable neuropeptides, enhanced signal to noise, increased spatial resolution, or a combination of these aspects. The second half of this review focuses on recent biological discoveries about neuropeptides resulting from these improvements in MSI analysis. The recent progress in neuropeptide detection and analysis methods, including the incorporation of various tissue washes, matrices, instruments, ionization sources, and computation approaches combined with the advancements in understanding neuropeptide function in a variety of model organisms, indicates the potential for the utilization of MSI analysis of neuropeptides in clinical settings.

神经肽是神经系统中一类重要的内源性肽，可调节进食、葡萄糖稳态、疼痛、记忆、生殖等生理功能。为了了解神经肽在疾病或病症中的功能作用，必须进行研究以丰度和定位的变化来调查其失调。由于多种神经肽被认为在每个生理过程中发挥功能性作用，因此需要能够同时对多种神经肽进行全局分析的技术。质谱分析非常适合实现这一目标，因为它能够在无需事先全面了解感兴趣的分析物的情况下执行非目标测量。质谱成像 (MSI) 特别有用，因为它能够在一次实验中对多种肽进行成像而无需标记。与所有分析技术一样，仔细的样品制备对于成功进行 MSI 分析至关重要。本综述的前半部分重点介绍了 MSI 样品制备和用于分析神经肽和其他生物分子的仪器的最新发展，其中样品制备技术可直接应用于神经肽分析。结合这些技术所带来的好处表现为许多可观察到的神经肽的改进、信噪比的增强、空间分辨率的增加或这些方面的组合。本综述的后半部分重点介绍了由于 MSI 分析的这些改进而产生的有关神经肽的最新生物学发现。 神经肽检测和分析方法的最新进展，包括各种组织洗涤、基质、仪器、电离源和计算方法的结合，以及对各种模型生物体中神经肽功能的理解的进展，表明了利用临床环境中神经肽的 MSI 分析。