Sialylation is a glycosylation feature that occurs in different linkages at the non-reducing end of a glycan moiety, the linkage isomers are often differentially associated with various biological processes. Due to very similar physico-chemical properties, the separation of isomeric sialylated glycopeptides remains challenging but of utmost importance in the biomedicine and biotechnology, including biomarker discovery, glyco-engineering and biopharmaceutical characterization. This study presents the implementation of a high-resolution separation platform based on capillary electrophoresis – mass spectrometry (CE–MS) allowing for the selective analysis of α2,3- and α2,6-sialylated glycopeptides. These differentially linked glycopeptides showed an identical fragmentation pattern (collision induced dissociation) but different electrophoretic mobilities, allowing for baseline separation of the different linkages without the need for an extensive sample preparation. The different migration behavior between the two moieties was found to correlate with differences in pK_avalues. Using a novel methodology adapted from the so-called internal standard CE approach, a relative difference of 3.4·10⁻²in pK_aunit was determined. This approach was applied for the analysis of tryptic glycopeptides of prostate specific antigen, which shows highly complex and heterogeneous glycosylation. The developed platform therefore appears attractive for the identification of differentially linked sialic acids that may be related to pathological conditions.

Metabolomics, which consists of the comprehensive analysis of metabolites within a biological system, has been playing a growing role in the implementation of personalized medicine in modern healthcare. A wide range of analytical approaches are used in metabolomics, notably mass spectrometry (MS) combined to liquid chromatography (LC), gas chromatography (GC), or capillary electrophoresis (CE). However, none of these methods enable a comprehensive analysis of the metabolome, due to its extreme complexity and the large differences in physico-chemical properties between metabolite classes. In this context, supercritical fluid chromatography (SFC) represents a promising alternative approach to improve the metabolome coverage, while further increasing the analysis throughput. SFC, which uses supercritical CO2 as mobile phase, leads to numerous advantages such as improved kinetic performance and lower environmental impact. This chromatographic technique has gained a significant interest since the introduction of advanced instrumentation, together with the introduction of dedicated interfaces for hyphenating SFC to MS. Moreover, new developments in SFC column chemistry (including sub-2 µm particles), as well as the use of large amounts of organic modifiers and additives in the CO2-based mobile phase, significantly extended the application range of SFC, enabling the simultaneous analysis of a large diversity of metabolites. Over the last years, several applications have been reported in metabolomics using SFC-MS - from lipophilic compounds, such as steroids and other lipids, to highly polar compounds, such as carbohydrates, amino acids, or nucleosides. With all these advantages, SFC-MS is promised to a bright future in the field of metabolomics.

Background: Succinate is produced by both host and microbiota, with a key role in the interplay of immunity and metabolism and an emerging role as a biomarker for inflammatory and metabolic disorders in middle-aged adults. The relationship between plasma succinate levels and cardiovascular disease (CVD) risk in young adults is unknown. Methods: Cross-sectional study in 100 (65% women) individuals aged 18–25 years from the ACTIvating Brown Adipose Tissue through Exercise (ACTIBATE) study cohort. CVD risk factors, body composition, dietary intake, basal metabolic rate, and cardiorespiratory fitness were assessed by routine methods. Plasma succinate was measured with an enzyme-based assay. Brown adipose tissue (BAT) was evaluated by positron emission tomography, and circulating oxylipins were assessed by targeted metabolomics. Fecal microbiota composition was analyzed in a sub-sample. Results: Individuals with higher succinate levels had higher levels of visceral adipose tissue (VAT) mass (+ 42.5%), triglycerides (+ 63.9%), C-reactive protein (+ 124.2%), diastolic blood pressure (+ 5.5%), and pro-inflammatory omega-6 oxylipins than individuals with lower succinate levels. Succinate levels were also higher in metabolically unhealthy individuals than in healthy overweight/obese peers. Succinate levels were not associated with BAT volume or activity or with fecal microbiota composition and diversity. Conclusions: Plasma succinate levels are linked to a specific pro-inflammatory omega-6 signature pattern and higher VAT levels, and seem to reflect the cardiovascular status of young adults.