Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
Anal Bioanal Chem. 2011 Oct;401(6):1949-61. doi: 10.1007/s00216-011-5249-z. Epub 2011 Jul 26.
There has been a recent surge in applications of mass spectrometry (MS) to tissue analysis, particularly lipid-based tissue imaging using ambient ionization techniques. This recent growth highlights the need to examine the effects of sample handling, storage conditions, and experimental protocols on the quality of the data obtained. Variables such as time before freezing after organ removal, storage time at -80 °C, time stored at room temperature, heating, and freeze/thaw cycles were investigated for their effect on the data quality obtained in desorption electrospray ionization (DESI)-MS using mouse brain. In addition, analytical variables such as tissue thickness, drying times, and instrumental conditions were also examined for their impact on DESI-MS data. While no immediate changes were noted in the DESI-MS lipid profiles of the mouse brain tissue after spending 1 h at room temperature when compared to being frozen immediately following removal, minor changes were noted between the tissue samples after 7 months of storage at -80 °C. In tissue sections stored at room temperature, degradation was noted in 24 h by the appearance of fatty acid dimers, which are indicative of high fatty acid concentrations, while in contrast, those sections stored at -80 °C for 7 months showed no significant degradation. Tissue sections were also subjected to up to six freeze/thaw cycles and showed increasing degradation following each cycle. In addition, tissue pieces were subjected to 50 °C temperatures and analyzed at specific time points. In as little as 2 h, degradation was observed in the form of increased fatty acid dimer formation, indicating that enzymatic processes forming free fatty acids were still active in the tissue. We have associated these dimers with high concentrations of free fatty acids present in the tissue during DESI-MS experiments. Analytical variables such as tissue thickness and time left to dry under nitrogen were also investigated, with no change in the resulting profiles at thickness from 10 to 25 μm and with optimal signal obtained after just 20 min in the dessicator. Experimental conditions such as source parameters, spray solvents, and sample surfaces are all shown to impact the quality of the data. Inter-section (relative standard deviation (%RSD), 0.44-7.2%) and intra-sample (%RSD, 4.0-8.0%) reproducibility data show the high quality information DESI-MS provides. Overall, the many variables investigated here showed DESI-MS to be a robust technique, with sample storage conditions having the most effect on the data obtained, and with unacceptable sample degradation occurring during room temperature storage.
近年来，质谱（MS）在组织分析中的应用越来越多，特别是使用环境电离技术的基于脂质的组织成像。这种最近的增长突出表明需要检查样品处理、储存条件和实验方案对获得的数据质量的影响。研究了器官切除后冷冻前的时间、-80°C 下的储存时间、室温下的储存时间、加热和冻融循环等变量对解吸电喷雾电离（DESI）-MS 中获得的数据质量的影响。此外，还研究了组织厚度、干燥时间和仪器条件等分析变量对 DESI-MS 数据的影响。尽管与立即冷冻相比，将小鼠脑组织在室温下放置 1 小时后，DESI-MS 脂质谱没有立即发生变化，但在 -80°C 下储存 7 个月后，组织样本之间存在较小的变化。在室温下储存的组织切片中，24 小时内通过脂肪酸二聚体的出现观察到降解，这表明脂肪酸浓度高，而相比之下，在 -80°C 下储存 7 个月的组织切片没有明显降解。组织切片还经历了多达六个冻融循环，并且每个循环后降解程度增加。此外，还将组织块置于 50°C 温度下，并在特定时间点进行分析。在短短 2 小时内，就观察到脂肪酸二聚体形成增加导致的降解，表明组织中形成游离脂肪酸的酶促过程仍在活跃。我们将这些二聚体与 DESI-MS 实验中组织中存在的高浓度游离脂肪酸联系起来。还研究了分析变量，如组织厚度和在氮气下干燥的时间，在 10 到 25μm 的厚度下，结果谱没有变化，在干燥器中仅 20 分钟后就获得了最佳信号。源参数、喷雾溶剂和样品表面等实验条件都显示出对数据质量的影响。交叉（相对标准偏差（%RSD），0.44-7.2%）和内样（%RSD，4.0-8.0%）重现性数据表明，DESI-MS 提供了高质量的信息。总体而言，这里研究的许多变量表明 DESI-MS 是一种稳健的技术，样品储存条件对获得的数据影响最大，并且在室温储存期间会发生不可接受的样品降解。
