Advanced Tandem Mass Spectrometric Analysis of Complex Mixtures of Triacylglycerol Regioisomers: A Case Study of Bovine Milk Fat.

Comprehensive analysis of triacylglycerol (TAG) regioisomers is extremely challenging, with many variables that can influence the results. Previously, we reported a novel algorithmic method for resolving regioisomers of complex mixtures of TAGs. In the current study, the TAG Analyzer software and its mass spectrometric fragmentation model were further developed and validated for a much wider range of TAGs. To demonstrate the method, we performed for the first time a comprehensive analysis of TAG regioisomers of bovine milk fat, a very important and one of the most complex TAG mixtures in nature containing FAs ranging from short to long carbon chains. This analysis method forms a solid basis for further investigation of TAG regioisomer profiles in various natural fats and oils, potentially aiding in the development of new and healthier foods and nutraceuticals with targeted lipid structures.

A novel UHPLC-ESI-MS/MS method and automatic calculation software for regiospecific analysis of triacylglycerols in natural fats and oils.

Regioisomeric analysis of triacylglycerols (TAGs) in natural oils and fats is a highly challenging task in analytical chemistry. Here we present a software (TAG Analyzer) for automatic calculation of regioisomeric composition of TAGs based on the mass spectral data from recently reported ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for analyzing TAG regioisomers. The software enables fast and accurate processing of complex product ion spectra containing structurally informative diacylglycerol [M+NH4-RCO2H-NH3]+ and fatty acid ketene [RCO]+ fragment ions. Compared to manual processing, the developed software offers higher throughput with faster calculation as well as more accurate interpretation of chromatographically overlapping isobaric TAGs. The software determines results by constructing a synthetic spectrum to match the measured fragment ion spectrum, and by reporting the optimal concentrations of TAGs used to create the synthetic spectrum. This type of calculation is often extremely challenging for manual interpretation of the fragment ion spectra of isobaric TAGs with shared fragments, hence the need for automated data processing. The developed software was validated by analyzing a wide range of mixtures of regiopure TAG reference compounds of known composition and a commercial olive oil sample. Additionally, the method was also applied for regiospecific analysis of TAGs in human milk as an example of natural fats and oils with a highly complex TAG profile. The results indicate that the software is capable of resolving regioisomeric composition of natural TAGs even of the most complex composition. This novel calculation software combined with our existing UHPLC-ESI-MS/MS method form a highly efficient tool for regioisomeric analysis of TAGs in natural fats and oils.

Preparation of Nitrogen Analogues of Ceramide and Studies of Their Aggregation in Sphingomyelin Bilayers.

Ceramides can regulate biological processes probably through the formation of laterally segregated and highly packed ceramide-rich domains in lipid bilayers. In the course of preparation of its analogues, we found that a hydrogen-bond-competent functional group in the C1 position is necessary to form ceramide-rich domains in lipid bilayers [Matsufuji; Langmuir 2018]. Hence, in the present study, we newly synthesized three ceramide analogues: CerN3, CerNH2, and CerNHAc, in which the 1-OH group of ceramide is substituted with a nitrogen functionality. CerNH2 and CerNHAc are capable of forming hydrogen bonds in their headgroups, whereas CerN3 is not. Fluorescent microscopy observation and differential scanning calorimetry analysis disclosed that these ceramide analogues formed ceramide-rich phases in sphingomyelin bilayers, although their thermal stability was slightly inferior to that of normal ceramides. Moreover, wide-angle X-ray diffraction analysis showed that the chain packing structure of ceramide-rich phases of CerNHAc and CerN3 was similar to that of normal ceramide, while the CerNH2-rich phase showed a slightly looser chain packing due to the formation of CerNH3+. Although the domain formation of CerN3 was unexpected because of the lack of hydrogen-bond capability in the headgroup, it may become a promising tool for investigating the mechanistic link between the ceramide-rich phase and the ceramide-related biological functions owing to its Raman activity and applicability to click chemistry.