Evaluation of Direct from Sample Metabolomics of Human Feces Using Rapid Evaporative Ionization Mass Spectrometry

Mass spectrometry
is a powerful tool in the investigation of the
human fecal metabolome. However, current approaches require time-consuming
sample preparation, chromatographic separations, and consequently
long analytical run times. Rapid evaporative ionization mass spectrometry
(REIMS) is a method of ambient ionization mass spectrometry and has
been utilized in the metabolic profiling of a diverse range of biological
materials, including human tissue, cell culture lines, and microorganisms.
Here, we describe the use of an automated, high-throughput REIMS robotic
platform for direct analysis of human feces. Through the analysis
of fecal samples from five healthy male participants, REIMS analytical
parameters were optimized and used to assess the chemical information
obtainable using REIMS. Within the fecal samples analyzed, bile acids,
including primary, secondary, and conjugate species, were identified,
and phospholipids of possible bacterial origin were detected. In addition,
the effect of storage conditions and consecutive freeze/thaw cycles
was determined. Within the REIMS mass spectra, the lower molecular
weight metabolites, such as fatty acids, were shown to be significantly
affected by storage conditions for prolonged periods at temperatures
above −80 °C and consecutive freeze/thaw cycles. However,
the complex lipid region was shown to be unaffected by these conditions.
A further cohort of 50 fecal samples, collected from patients undergoing
bariatric surgery, were analyzed using the optimized REIMS parameters
and the complex lipid region mass spectra used for multivariate modeling.
This analysis showed a predicted separation between pre- and post-surgery
specimens, suggesting that REIMS analysis can detect biological differences,
such as microbiome-level differences, which have traditionally been
reliant upon methods utilizing extensive sample preparations and chromatographic
separations and/or DNA sequencing.

质谱（Mass spectrometry）是探究人类粪便代谢组的强有力工具。然而，现有分析方法需耗费大量时间完成样品前处理与色谱分离，导致分析运行周期较长。快速蒸发电离质谱（Rapid evaporative ionization mass spectrometry, REIMS）属于环境电离质谱技术范畴，已被应用于多种生物样本的代谢谱分析，涵盖人体组织、细胞培养系及微生物等。本研究介绍了一套自动化高通量REIMS机器人平台，用于人类粪便的直接分析。通过对5名健康男性志愿者的粪便样本进行分析，本研究优化了REIMS的分析参数，并以此评估了REIMS可获取的化学信息。在所分析的粪便样本中，研究人员鉴定出包括初级、次级及结合型胆汁酸在内的多种胆汁酸，并检测到可能源自细菌的磷脂。此外，本研究还考察了储存条件与反复冻融循环的影响。在REIMS质谱图中，分子量较低的代谢物（如脂肪酸）在-80℃以上环境长期储存及反复冻融循环后，信号会受到显著影响。但复合脂类区域的信号则不受上述因素影响。本研究进一步纳入50名接受减重手术患者的粪便样本队列，采用优化后的REIMS参数进行分析，并以复合脂类区域的质谱数据开展多变量建模。该分析结果显示手术前后的样本可实现有效区分，这表明REIMS分析能够检测到传统上需依赖复杂样品前处理、色谱分离及/或DNA测序技术才能发现的生物学差异（如微生物组层面的差异）。