Goat Colostrum Oligosaccharides Characterization by Nano-LC-Chip-Q-TOF MS and HILIC-MS

Title: Characterization of Goat Colostrum Oligosaccharides by Nanoliquid Chromatography on Chip Quadrupole Time-of-Flight Mass Spectrometry and Hydrophilic Interaction Liquid Chromatography-Quadrupole Mass Spectrometry

Journal: Journal of chromatography A

Published: 2016

Background

Goat milk is a nutritionally rich source containing bioactive components like carbohydrates, lipids, and proteins. Although lactose is the primary carbohydrate, goat milk also contains oligosaccharides (OS) with structures similar to those found in human milk. These oligosaccharides are classified into neutral and acidic types, with bioactive properties such as prebiotic effects and pathogen-binding activities. While significant research has focused on human milk oligosaccharides, limited data is available on goat milk OS, particularly in colostrum, which is expected to have higher OS content than mature milk. Advanced analytical techniques, such as nanoliquid chromatography (Nano-LC) and hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (MS), offer sensitive and detailed characterization of these compounds. This study aims to comprehensively characterize and quantify goat colostrum oligosaccharides (GCO) using Nano-LC-Chip-Q-TOF MS and HILIC-MS.

Materials & Methods

Chemicals and Reagents:

All reagents used were of analytical grade or higher. Various solvents like acetic acid, ammonium acetate, ammonium hydroxide, ethanol, and acetonitrile were sourced from reputable suppliers. Analytical standards of oligosaccharides, including β-4-galactosyl-lactose, maltotriose, and 6′-sialyl-lactose, were used for analysis.

Colostrum Samples:

Colostrum samples were collected from Murciano-Granadina goats from two farms in Granada, Spain. Samples were stored at −80°C. Ethical approval was obtained, and animals were handled according to Spanish and European guidelines.

Fat and Protein Removal:

Samples were defatted via centrifugation and filtration. Proteins were precipitated using ethanol, and the carbohydrate fraction was extracted, evaporated, and lyophilized.

Oligosaccharide Isolation:

Size-exclusion chromatography (SEC) was used to remove mono- and disaccharides, enriching the oligosaccharide fraction. Mass spectrometry (ESI-MS) confirmed the degree of polymerization of fractions with DP ≥ 3.

Chromatographic Analyses:

Qualitative Analysis: Nano-LC-Chip-Q-TOF MS was used for the qualitative identification of oligosaccharides. Separation was achieved using a graphitized carbon column with a binary gradient of acetonitrile and formic acid. MS/MS analysis confirmed oligosaccharide compositions.
Quantitative Analysis: HILIC-QMS was performed using a hybrid column. Positive and negative electrospray ionization modes were applied for detection. Calibration curves were prepared for quantification using external standards, ensuring reproducibility and method validation.

Results

Qualitative Analysis of Goat Colostrum Oligosaccharides (GCO)

Using Nano-LC-Chip-Q-TOF MS, a total of 78 oligosaccharides (OS) were identified from goat colostrum samples, with 59 confirmed through MS/MS analysis.
GCO exhibited greater structural complexity compared to human or bovine milk oligosaccharides.
The purification process using SEC successfully reduced lactose content by 99.9%, though some loss of neutral OS with three monomeric units occurred.
The OS identified included:
- Neutral Non-Fucosylated OS (40, 51.3%)
- Neutral Fucosylated OS (3, 3.8%)
- Sialylated OS (35, 44.9%), containing N-acetylneuraminic (Neu5Ac) or N-glycolylneuraminic (Neu5Gc) acids.
Sialyl-lactoses were the most prevalent, followed by Hex-HexNAc-Neu5Ac and Hex-HexNAc-Neu5Gc.
Higher Neu5Gc content (54.8%) was found compared to previous studies (29.4%).
Three fucosylated OS were identified, although low abundance limited MS/MS confirmation.

Quantitative Analysis of GCO

HILIC-Q MS was optimized using a representative colostrum sample (CS5). Different chromatographic conditions were tested, with 0.1% ammonium hydroxide providing the best separation.
Acidic OS eluted earlier than neutral OS under basic conditions. Positive polarity yielded better detection for all OS types compared to negative polarity.
The analysis showed the following results across five colostrum samples:
- Total OS concentrations ranged from 251.22 to 572.24 mg/L.
- Neutral OS concentrations varied from 140 to 315 mg/L.
- Acidic OS concentrations ranged from 83 to 251 mg/L.
Galactosyl-lactoses were the most abundant OS, with concentrations between 124.92 and 265.77 mg/L.
Among acidic OS, 6′-SL concentrations (28.85–123.76 mg/L) were consistently higher than 3′-SL (3.05–11.99 mg/L), resembling human milk more than bovine milk.
Fucosyl-oligosaccharides such as 2′-FL (2.21–31.59 mg/L) and fucosyl-lactosamine (3.08–6.15 mg/L) were also detected.
Sialyl-lactosamine isomers showed notable presence, ranging from 5.17 to 65.12 mg/L.

Nano-LC-Chip-Q-TOF MS profiles of goat colostrum oligosaccharides: CS1, CS2, CS3, CS4, and CS5

MS/MS spectra of: (a) hexosyl-lactose 3_0_0_0_0 (m/z 505.176), (b) 2′-FL (m/z 489.181), and (c) 3′-SL (634.218).

Extracted ion chromatographic profile of CS5 oligosaccharides obtained using BEH amide column and 0.1% ammonium hydroxide as mobile phase additive.

Reference

Martín-Ortiz, Andrea, et al. "Characterization of goat colostrum oligosaccharides by nano-liquid chromatography on chip quadrupole time-of-flight mass spectrometry and hydrophilic interaction liquid chromatography-quadrupole mass spectrometry." Journal of chromatography A 1428 (2016): 143-153. http://dx.doi.org/10.1016/j.chroma.2015.09.060

* For Research Use Only. Not for use in diagnostic procedures.

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