8 research outputs found

    Measured relative abundances from a single injection of OS standards labeled in a 2:1 ratio.

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    <p>Measured relative abundances from a single injection of OS standards labeled in a 2:1 ratio.</p

    Comparison of four high-molecular weight fucosylated OS abundances in Holstein (H) and Jersey (J) milk.

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    <p>Error bars show mean ± standard deviation. OS compositions are expressed as Hex_NexNAc_Fuc_NeuAc_NeuGc. Asterisks represent statistical significance from a two-tailed unpaired t-test at p<0.05 (*) and p<0.01 (**).</p

    Annotated total ion chromatogram of aminoxyTMT-labeled bovine milk OS.

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    <p>OS are represented by their monosaccharide compositions, denoted as Hex_HexNAc_Fuc_NeuAc_NeuGc. 3’-SL, 3’-sialyllactose; 6’-SL, 6’-sialyllactose; LNH, lacto-N-hexaose.</p

    Tandem mass tag structure and labeling reaction.

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    <p>Using collision-induced dissociation, tags fragment as shown by the blue line above.</p

    Multiplexed bovine milk oligosaccharide analysis with aminoxy tandem mass tags

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    <div><p>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.</p></div

    Ionization efficiency improvement following aminoxyTMT labeling.

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    <p>Injection of identical sample quantities before and after labeling resulted in increased signal intensity for numerous OS, as shown in the ESI mass spectra above for the OS of composition 3 Hex 6 HexNAc 1 Fuc (A–native OS; B–derivatized OS). Ions containing the aminoxyTMT label are annotated in blue; those without the label are shown in red. In-source fragments are annotated in green. OS are represented by their monosaccharide compositions, denoted as Hex_HexNAc_Fuc_NeuAc_NeuGc. 3’-SL, 3’-sialyllactose.</p

    Measured relative abundances of 15 milk oligosaccharides identified from a single sample, labeled with two isobaric label variants in a 2:1 ratio.

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    <p>The listed ratios represent the average of four repeat injections of the same sample. Oligosaccharides are represented by their monosaccharide compositions, denoted as Hex_HexNAc_Fuc_NeuAc_NeuGc.</p

    MS/MS spectrum of lacto-N-tetraose in profile (upper) and centroid (lower) formats.

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    <p>Resolution, defined as the quotient of m/z and peak width, or m/Δm, of the TMT<sup>6</sup>-126 reporter ion and the HexNAc oxonium ion at m/z 126.055 are sufficient for successful differentiation of the two peaks. The instrument provides near baseline separation of the ions in the profile data and allows successful conversion to centroid format.</p