Abstract
Milk oligosaccharides (OS) are a key factor that influences the infant gut microbial composition, and their importance in promoting healthy infant development and disease prevention is becoming increasingly apparent. Investigating the structures, properties, and sources of these compounds requires a host of complementary analytical techniques. Relative compound quantification by mass spectral analysis of isobarically labeled samples is a relatively new technique that has been used mainly in the proteomics field. Glycomics applications have so far focused on analysis of protein-linked glycans, while analysis of free milk OS has previously been conducted only on analytical standards. In this paper, we extend the use of isobaric glycan tags to the analysis of bovine milk OS by presenting a method for separation of labeled OS on a porous graphitized carbon liquid chromatographic column with subsequent analysis by quadrupole time-of-flight tandem mass spectrometry. Abundances for 15 OS extracted from mature bovine milk were measured, with replicate injections providing coefficients of variation below 15% for most OS. Isobaric labeling improved ionization efficiency for low-abundance, high-molecular weight fucosylated OS, which are known to exist in bovine milk but have been only sporadically reported in the literature. We compared the abundances of four fucosylated OS in milk from Holstein and Jersey cattle and found that three of the compounds were more abundant in Jersey milk, which is in general agreement with a previous study. This novel method represents an advancement in our ability to characterize milk OS and provides the advantages associated with isobaric labeling, including reduced instrumental analysis time and increased analyte ionization efficiency. This improved ability to measure differences in bioactive OS abundances in large datasets will facilitate exploration of OS from all food sources for the purpose of developing health-guiding products for infants, immune-compromised elderly, and the population at large.
Highlights
Milk oligosaccharides (OS) have been intensely researched in the past two decades due to their multitude of bioactivities
A mixture of four OS normally found in bovine milk (3’-SL, 6’-SL, lacto-N-tetraose, and lactoN-hexaose) [15, 38] was labeled in a 2:1 ratio with the TMT6-126 and TMT6-127 reagents
Upon liquid chromatography (LC)-mass spectrometry (MS)/MS analysis, the electrospray ionization (ESI) mass spectra showed that the OS standards ionized as hydrogen adducts with a mixture of 1+ and 2+ charges, as shown in Fig 2 for lacto-N-tetraose and lacto-N-hexaose
Summary
Milk oligosaccharides (OS) have been intensely researched in the past two decades due to their multitude of bioactivities. Experimental evidence demonstrates that milk OS can act as prebiotics [1,2,3], prevent severe diarrhea [4, 5] and necrotizing entercolitis in infants [6], modulate. Bovine milk oligosaccharide analysis with tandem mass tags
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