Multiplexed bovine milk oligosaccharide analysis with aminoxy tandem mass tags

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.

Profiling of aminoxyTMT-labeled bovine milk oligosaccharides reveals substantial variation in oligosaccharide abundance between dairy cattle breeds

“…Oligosaccharides were extracted and labeled with isobaric reagents as described previously 33 . Briefly, milk aliquots of 400 µL were combined with an equal volume of Milli-Q water and skimmed at 4,000 × g and 4 °C for 30 min.…”

“…Aliquots of 7.5% of each sample extract were labeled with aminoxy tandem mass tag (TMT) reagents using the manufacturer’s protocol (ThermoFisher Scientific, Waltham, MA). Since each multiplexed set has a maximum of six available reporter ion channels and the study included over 600 total samples, the samples were multiplexed using a recently-published strategy 33 in which each multiplexed vial contains five TMT-labeled samples and one TMT-labeled OS standard mixture that is used to normalize the TMT reporter ion intensities. The bovine milk OS powder used as the standard mixture was produced according to processing techniques described previously 35 and was extracted using the C18 and PGC SPE procedures described above.…”

“…Chromatographic separation was performed on a PGC high-performance LC chip, which contained a 40 nL loading column and a 75 µm × 43 mm analytical column, both packed with 5 µm particles. Instrumental parameters for chromatographic separation and OS analysis by MS have been described previously 33 . Ionization source voltage was varied from 1825–1890 V to maintain a stable solvent spray.…”

“…Given that Holstein-Friesian and Jersey are two of the most commonly used breeds for milk production, we have designed a comprehensive study to further investigate the milk OS content of these cattle breeds to better understand how factors such as breed and milk yield may influence variation in the milk OS profile. We have measured relative quantities of OS in a large collection of milk samples using LC-MS and isobaric labeling, an analytical technique that improves instrumental throughput for large sample sets by allowing samples to be multiplexed prior to analysis by mass spectrometry 33 . Building our understanding of variables that affect OS abundances in milk and its processing side-products will enable OS to be recovered more efficiently from industrial processing streams for use in infant formulas and therapeutic foods for children and adults.…”

Evaluation of 6′-Sialyllactose Sodium Salt Supplementation to Formula on Growth and Clinical Parameters in Neonatal Piglets

“…A total of 30-50 bovine milk oligosaccharides (BMO) structures have been reported in bovine milk and colostrum [7]. Human and bovine milk share at least 10 common structures, of which 3 -SL and 6 -SL comprise a largest percentage of the BMO [7,8]. Earlier reports quantified the levels of oligosaccharide bound sialic acid in mature bovine milk at 33 mg/L and unsupplemented infant formula at 38 mg/L [9].…”