Microbiota independent effects of oligosaccharides on Caco-2 cells -A semi-targeted metabolomics approach using DI-FT-ICR-MS coupled with pathway enrichment analysis

Abstract

Milk oligosaccharides (MOS) and galactooligosaccharides (GOS) are associated with many benefits, including anti-microbial effects and immune-modulating properties. However, the cellular mechanisms of these are largely unknown. In this study, the effects of enriched GOS and MOS mixtures from caprine and bovine milk consisting mainly 6'-galactosyllactose, 3'-sialyllactose, and 6'-sialyllactose on Caco-2 cells were investigated, and the treatment-specific metabolomes were described. In the control, the cells were treated with a sugar mix consisting of one-third each of glucose, galactose and lactose.A local metabolomics workflow with pathway enrichment was established, which specifically addresses DI-FT-ICR-MS analyses and includes adaptations in terms of measurement technology and sample matrices. By including quality parameters, especially the isotope pattern, we increased the precision of annotation. The independence from online tools, the fast adaptability to changes in databases, and the specific adjustment to the measurement technology and biomaterial used, proved to be a great advantage.For the first time it was possible to find 71 active pathways in a Caco-2 cell experiment. These pathways were assigned to 12 main categories, with amino acid metabolism and carbohydrate metabolism being the most dominant categories in terms of the number of metabolites and metabolic pathways. Treatment of Caco-2 cells with high GOS and glucose contents resulted in significant effects on several metabolic pathways, whereas the MOS containing treatments resulted only for individual metabolites in significant changes. An effect based on bovine or caprine origin alone could not be observed. Thus, it was shown that MOS and GOS containing treatments can exert microbiome-independent effects on the metabolome of Caco-2 cells.

New perspectives on ‘Breathomics’: metabolomic profiling of non-volatile organic compounds in exhaled breath using DI-FT-ICR-MS

“…To obtain QCs, 10 µl of each sample were transferred to a collective sample vial and subsequently processed identically compared to the analytical samples. The QC sample was measured in the beginning, after 12 and 24 samples, respectively, and at the end of each method following a modified approach of Jensen-Kroll et al 75 . In total, 34 QC samples were measured for each method.…”

Simultaneous Quantitation and Discovery (SQUAD) Analysis: Combining the Best of Targeted and Untargeted Mass Spectrometry-Based Metabolomics

“…In this protocol, the semi-targeted term was used to describe the level of identification, which benefited from using NMR, rather than increased metabolite recovery [ 54 ]. Similarly, other analyses were done for the confident annotation of lipids using HRAM [ 55 ], QTRAP [ 56 ], QTOF [ 57 , 58 ], or metabolites using DI-FT-ICR-MS coupled with pathway enrichment analysis [ 59 ], but authors still called these approaches semi-targeted lipidomics or metabolomics. In another example, MS 2 spectra of metabolites resulting from a QTRAP instrument were compared with references MS 2 spectra for the confident annotation of potential biomarkers of fertility decline in elderly women, which might help in finding the corresponding traditional Chinese medicine treatment and exploring its mechanism of action [ 60 ].…”

Simultaneous Quantitation and Discovery (SQUAD) Analysis: Combining the Best of Targeted and Untargeted Mass Spectrometry-Based Metabolomics

“…In this protocol, the semi-targeted term was used to describe the level of identification, which benefited from using NMR, rather than increased metabolite recovery [80]. Similarly, other analyses were done for the confident annotation of lipids using HRAM [81], QTRAP [82], QTOF [83,84], or metabolites using DI-FT-ICR-MS coupled with pathway enrichment analysis [85] but still calling these approaches semi-targeted lipidomics or metabolomics. In another example, MS 2 spectra of metabolites resulting from a QTRAP instrument were compared with references MS 2 spectra for the confident annotation of potential biomarkers of fertility decline in elderly women, which might help in finding the corresponding traditional Chinese medicine treatment and exploring its mechanism of action [86].…”