Session: Session 1
Characterization of porcine and rabbit milk oligosaccharides by liquid chromatography coupled to high-resolution mass spectrometry
Introduction
Milk provides newborns with essential nutrients for the development of the intestinal microbiota and the immune system. Indeed, milk oligosaccharides (MOs) act as bioactive components conferring benefits to the early development of infant gut microbiota. MOs are particularly difficult to analyze because of the numerous coexisting isomeric structures and multiple connectivity sites, rendering their separation and detailed structural characterization difficult to obtain. Here we report the development of an analytical strategy based on liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) for analyzing MOs present in the colostrum and milk samples of pigs and rabbits.
Methodology
Colostrum and milk samples were first delipidated by centrifugation at 4°C during 30 minutes and then with a mixture of chloroform/methanol. The supernatant was then recovered and deproteinized by ethanol precipitation. MOs were reduced with sodium borohydride and purified by porous graphitic carbon columns before being analyzed by liquid chromatography coupled to a quadrupole-time-of-flight mass spectrometer (Q-TOF Impact HD, Bruker). Monosaccharide compositions of MOs were deduced from accurately measured masses and MS/MS experiments. Data were processed using the Skyline software.
Results and Conclusion
More than 50 MOs were identified in the few porcine colostrum and milk samples studied. Sialylated MOs were the predominant species, comprising more than 65% of the detected structures. Rabbit MOs were also majoritarily acidic with up to 75% of the ~30 species detected in colostrum being sialylated. Method reproducibility was evaluated on the colostrum samples of both origins and showed CV below 15% on average for MOs whose relative abundance > 1%. Preliminary results showed that relative abundances of sialylated MOs tend to decrease over the course of lactation for both mammalian species. Additional experiments are on-going to confirm this assumption.
Milk provides newborns with essential nutrients for the development of the intestinal microbiota and the immune system. Indeed, milk oligosaccharides (MOs) act as bioactive components conferring benefits to the early development of infant gut microbiota. MOs are particularly difficult to analyze because of the numerous coexisting isomeric structures and multiple connectivity sites, rendering their separation and detailed structural characterization difficult to obtain. Here we report the development of an analytical strategy based on liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) for analyzing MOs present in the colostrum and milk samples of pigs and rabbits.
Methodology
Colostrum and milk samples were first delipidated by centrifugation at 4°C during 30 minutes and then with a mixture of chloroform/methanol. The supernatant was then recovered and deproteinized by ethanol precipitation. MOs were reduced with sodium borohydride and purified by porous graphitic carbon columns before being analyzed by liquid chromatography coupled to a quadrupole-time-of-flight mass spectrometer (Q-TOF Impact HD, Bruker). Monosaccharide compositions of MOs were deduced from accurately measured masses and MS/MS experiments. Data were processed using the Skyline software.
Results and Conclusion
More than 50 MOs were identified in the few porcine colostrum and milk samples studied. Sialylated MOs were the predominant species, comprising more than 65% of the detected structures. Rabbit MOs were also majoritarily acidic with up to 75% of the ~30 species detected in colostrum being sialylated. Method reproducibility was evaluated on the colostrum samples of both origins and showed CV below 15% on average for MOs whose relative abundance > 1%. Preliminary results showed that relative abundances of sialylated MOs tend to decrease over the course of lactation for both mammalian species. Additional experiments are on-going to confirm this assumption.