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Changes in caprine milk oligosaccharides at different lactation stages analyzed by high performance liquid chromatography coupled to mass spectrometry Andrea Martin-Ortiz, Daniela Barile, Jaime Salcedo, F Javier Moreno, Alfonso Clemente, ANA ISABEL RUIZ MATUTE, and Maria Luz Sanz J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05104 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Changes in caprine milk oligosaccharides at different lactation stages analyzed by

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high performance liquid chromatography coupled to mass spectrometry

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Andrea Martín-Ortiz1, Daniela Barile2, Jaime Salcedo2, F. Javier Moreno3, Alfonso

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Clemente4, Ana I. Ruiz-Matute1*, María L. Sanz1

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(Spain)

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Shields Avenue, Davis, CA 95616, USA

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Instituto de Química Orgánica General (CSIC). Juan de la Cierva, 3 28006 Madrid,

Department of Food Science and Technology, University of California Davis, One

Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), C/

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Nicolás Cabrera, 9, Campus de Cantoblanco - Universidad Autónoma de Madrid, 28049

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Madrid, Spain

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(CSIC), Profesor Albareda 1, 18008 Granada, Spain

Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas

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*Corresponding author:

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Email: [email protected]

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Tel. +34915622900

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Fax: +34925644853

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Abstract

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Changes of the abundance of caprine milk oligosaccharides (CMO) at different lactation

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stages has been evaluated by hydrophilic interaction liquid chromatography coupled to

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mass spectrometry (HILIC-Q MS) and nano-flow liquid chromatography-quadrupole-

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time of flight mass spectrometry (nano-LC-Chip-QTOF MS). Eight major

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oligosaccharides (OS) were quantified at different lactation stages by HILIC-Q MS,

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whilst the use of nano-LC-Chip-QToF MS allowed expanding the study to forty-nine

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different OS by monitoring neutral non- and fucosylated species, as well as acidic

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species containing not only N-acetyl-neuraminic acid or N-glycolyl-neuraminic acid

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residues but also the combination of both sialic acids. Overall, the most abundant OS

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decreased with lactation time, whereas different trends were observed for minor

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oligosaccharides. 6’-Sialyl-lactose was the most abundant acidic OS whilst galactosyl-

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lactose isomers were identified as the most abundant neutral OS. This is the first time

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that a comprehensive study regarding the changes of the abundance of CMO, both

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neutral and acidic, at different lactation stages is carried out.

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Keywords: goat milk oligosaccharides, lactation stages, hydrophilic interaction liquid

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chromatography, nanoflow liquid chromatography-quadrupole-time of flight mass

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spectrometry

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Journal of Agricultural and Food Chemistry

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Introduction

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Human milk (HM) is considered the ideal food for neonates, not only because of its

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complex nutrients mixture fulfilling infant’s requirements but also because of the

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presence of bioactive components exerting important physiological effects for the

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neonate. 1 Among those, human milk oligosaccharides (HMOs) have been described to

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act as prebiotics and as potent inhibitors of bacterial adhesion to intestinal epithelial

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receptors.

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proportion in the gastrointestinal tract, they may play a role in the local intestinal

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immune system of the breast-fed infants, thus, acting in the prevention of infectious

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diseases. 2-8

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HMOs are complex structures with different glycosidic linkages, degrees of

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polymerization and monomeric composition including hexoses (Hex, D-glucose and D-

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galactose), N-acetyl-glucosamine (GlcNAc), L-fucose (Fuc), and sialic acid [N-acetyl-

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neuraminic acid (Neu5Ac)].

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present in HM,

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to acidic ones (10-25%).

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diversity, at present, it is not feasible to totally replicate HMOs profile by synthetic

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methods; only HMOs of lower molecular weight have been successfully produced by

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molecular engineering approaches.

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alternative sources with similar bioactive properties to HMOs that can be exploited by

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the food or pharmaceutical industry on a large scale, either through direct consumption

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or as a functional ingredient in infant formulas, mimicking biological functions of

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oligosaccharides present in HM. 15, 16 In this sense, naturally-occurring OS isolated from

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milk of animal species could be considered. 17, 18

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Moreover, as they are not digested and absorbed only in a small

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More than 200 different OS have been described to be

with predominance of neutral fucosylated OS (50-70%) compared 12, 13

However, due to their specific structural composition

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Therefore, there is great interest in finding

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Goat milk is known to contain oligosaccharides structurally similar to those present in

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HM

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Recently, in vitro studies have revealed the potential prebiotic activity of

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oligosaccharides from caprine whey.

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oligosaccharides isolated from goat milk reduce intestinal inflammation in rats with

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induced colitis

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Therefore, goat milk could be a potentially promising functional food ingredient for

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improving intestinal health, 24 especially if supplemented in infant formulae.

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Liquid chromatography (LC) is the most widespread technique for the analysis of

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oligosaccharides from mammal milks. Although several LC operation modes such as

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normal phase (NP) 25 and high performance anion exchange chromatography (HPAEC)

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4, 14, 22, 23, 26, 27

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interaction liquid chromatography (HILIC) is considered an efficient operation mode for

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the analysis of complex OS mixtures

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isomeric oligosaccharide and good peak shapes. In a recent study from our group, 20 the

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main OS in different goat colostra have been quantified by HILIC-Q MS. Additionally,

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up to 78 caprine milk oligosaccharides (CMO) structures were identified by nanoflow

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liquid chromatography-quadrupole-time of flight mass spectrometry (Nano-LC-Chip–

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Q-TOF MS). Combination of information provided by both techniques allowed

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obtaining comprehensive information, not provided in previous studies, about goat

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colostra oligosaccharide composition.

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Changes of the oligosaccharide abundance of human and bovine milks at different

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lactation stages have been widely studied, observing a decrease in their total content

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during lactation and a modification in the OS profile.

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about the changes of CMO composition during lactation. A transcriptomic study of

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and in higher concentrations than milks from other farm mammals.

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Moreover, it has been proved that

and they can improve barrier function of epithelial cell co-cultures.

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have been widely used for the analysis of these carbohydrates, hydrophilic

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providing appropriate resolution between

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However, little is known

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Journal of Agricultural and Food Chemistry

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glycosylation-related genes in goat colostrum and milk obtained after 120 days of

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lactation has recently shown that most of them were more expressed in the former,

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which might be related to the higher oligosaccharides concentration found in colostrum.

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(6’-SL) and disialyl-lactose in colostra and milks of two goat breeds sampled at

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different stages of lactation (up to 90 days) was reported. 27, 35 In general, a decrease of

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OS content was observed during lactation, although 3’-SL contents increased in

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colostrum from 0 h to 24 h. Formiga de Sousa et al. 36 also detected a decrease in sialic

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acid monomers, obtained after hydrolysis of CMO, at different lactation stages (85, 105,

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125 and 145 days). However, to the best of our knowledge, a more comprehensive study

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of the evolution of both neutral and acidic CMO during lactation has not yet been

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carried out.

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Therefore, the present work investigated the changes of the abundance of CMO during

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the first months of lactation in individual and pooled milks from goats of the same breed

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(Murciano-Granadina) by HILIC-Q MS and Nano-LC-Chip–Q-TOF MS.

Recently, the content of three OS, namely 3’-sialyl-lactose (3’-SL), 6’-sialyl-lactose

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Materials and Methods

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Chemicals and reagents

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Acetic acid was from Normasolv (Barcelona, Spain); ammonium acetate and

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ammonium hydroxide were from Panreac (Barcelona, Spain), and ethanol of analytical

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grade was acquired from Lab-Scan (Gliwice, Poland). Acetonitrile (ACN) and formic

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acid (HPLC-MS grade) were obtained from Fisher-Scientific (Fair Lawn, NJ, USA).

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ESI-TOF Low concentration Tuning Mix G1969–85000 was acquired from Agilent

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Technologies (Santa Clara, CA, USA). Nanopure water (18.2 MΩ.cm, 25 °C) was used

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throughout all experiments.

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Analytical grade standards of 6’-sialyllactose (6’-SL) sodium salt, 3’-sialyllactose (3’-

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SL) sodium salt, 3’-sialyl-N-acetyllactosamine, 3’-galactosyl-lactose, 4’-galactosyl-

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lactose and 6’-galactosyl-lactose were purchased from Carbosynth (Berkshire, UK),

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whereas 3α,4β-galactotriose was obtained from Dextra Laboratories (Reading, UK) and

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2’-fucosyllactose (2’-FL) was from V-Labs (Covington, LA, USA). Nylon FH

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membranes (0.22 µm; Millipore, Bedford, MA, USA) were used for filtering

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ACN:water (50:50, v:v) standard solutions before injection.

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Milk samples

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Individual milk samples from one Murciano-Granadina goat (GM) were collected at an

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experimental farm at Estación Experimental del Zaidín (Granada, Spain). Samples were

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collected at days 2, 10, 20, 30 and 40 of lactation (GM2, GM10, GM20, GM30 and

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GM40, respectively). Moreover, milks kindly provided by Hermanos Archiduque farm

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(Granada, Spain) from twelve individual Murciano-Granadina goats (GP) were

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collected at days 1, 7, 30 and 120 and pooled (GP1, GP7, GP30 and GP120,

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respectively). GM2 and GP1 samples were considered colostrum. Samples were

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immediately frozen at -80 °C until their analysis. The Spanish guidelines for

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experimental animal protection (Royal Decree 53/2013) in line with the corresponding

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European Directive (2010/63/EU) were followed animals care and handle. The Ethics

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Committee for Animal Research from the Animal Nutrition Unit approved the

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experimental protocol.

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Fat and protein removal

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Samples were defatted by centrifugation at 6500 × g for 15 min at 5 ºC and kept in an

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ice bath for 30 min. Whatman Nº 1 filter paper was used for the removal of lipid layer

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supernatant.4

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The removal of the protein fraction was carried out by the addition of two volumes of

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cold ethanol to the skimmed colostrum and milk samples. After shaking for 2 h in an ice

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bath, solutions were centrifuged at 6500 × g for 30 min at 5 ºC. Supernatants were

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collected and ethanol was removed ple in a rotary evaporator (Büchi Labortechnik AG,

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Flawil, Switzerland) at 37 ºC. The carbohydrate fraction present in the aqueous solution

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was frozen and freeze-dried.

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Chromatographic analyses

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HILIC-Q MS

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CMO quantification was performed on an Agilent 1200 series HPLC system (Hewlett-

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Packard, Palo Alto, CA, USA) equipped with an oven (Kariba Instruments, UK) and

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coupled to a quadrupole HP-1100 mass detector (Hewlett-Packard) with an electrospray

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ionization (ESI) source using the method previously optimized by Martín-Ortiz et al. 20.

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Samples were diluted with 50:50 (v:v) acetonitrile (ACN):water, filtered through a 0.22

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µm filter (Millipore, Madrid , Spain) and 5 µL were injected.

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An ethylene bridge hybrid with trifunctionally-bonded amide phase (BEH X-Bridge

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column; 150 x 4.6 mm; 3.5 m particle size, 135 Å pore size, Waters, Hertfordshire, UK)

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at a flow rate of 0.4 mL min-1 was used for LC experiments. For these assays, a gradient

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of ACN:water with addition of 0.1% ammonium hydroxide was used as follows: from 0

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to 46 min a linear gradient from 15 to 50% aqueous phase, kept 10 min at these

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conditions, then ramped to original composition in 1 min and finally equilibrated for 15

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min. The ESI source was operated under positive polarity using 4 kV capillary voltage

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at 300 ºC with a nitrogen drying gas flow of 12 L min−1 and a nebulizer (N2, 99.5%

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purity) pressure of 276 kPa; fragmentor voltage was varied from 80 to110 V. Adducts

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formed under optimal conditions were evaluated. Ions corresponding to [M+Na]+ of the

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oligosaccharides under analysis were monitored in SIM mode using default variable

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fragmentor voltages. HPChem Station software version 10.02 (Hewlett-Packard) was

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used for data processing.

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Quantitative analysis was performed in triplicate by the external standard method, using

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calibration curves within the range 5-100 mg L-1 for 3’-SL, 6’-SL, 3’-sialyl-N-acetyl-

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lactosamine and 3’-galactosyllactose. Considering the absence of glycoly-neuraminyl-

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lactose (Neu5Gc-lactose) as commercial standard, the responses of N-acetyl-neuraminic

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and N-glycolyl-neuraminic acids were evaluated. Negligible differences were observed

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between the responses of these monosaccharides; therefore, Neu5Gc-lactose isomers

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were quantified using 3’-SL calibration curves. Reproducibility of the method was

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estimated on the basis of the intra-day and inter-day precision, calculated as the relative

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standard deviation (RSD) of retention times and concentrations of oligosaccharide

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standards obtained in five independent measurements. Good precision values were

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obtained (RSD ranging 6–8%). Limit of detection (LOD) and limit of quantitation

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(LOQ) were calculated as three and ten times, respectively, the signal to standard

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deviation of the noise ratio (S/σΝ). Values ranging from 0.03 - 0.16 mg mL−1 and 0.1 -

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0.5 mg mL−1 were obtained for LOD and LOQ, respectively.

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Nano-LC-Chip–Q-TOF MS -

Sample preparation

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Considering that the high content of lactose present in goat milk would cause

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interference with the analysis of lower abundance oligosaccharides, this disaccharide

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was removed from milk samples by size exclusion chromatography (SEC) using a Bio-

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Gel P2 (Bio-Rad, Hercules, CA, USA) column (90 × 5 cm). Water was selected as the

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mobile phase at a flow of 1.5 mL min-1 at 4 ºC. Oligosaccharide fraction was dissolved

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in water (20% wt:v) and 25 mL was injected into the column. Fractions of 10 mL were

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collected. The degree of polymerization (DP) of collected fractions was determined by

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ESI-MS (Agilent 1200 series HPLC system, Hewlett-Packard) at positive polarity

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selecting the corresponding m/z values. Fractions with DP ≥3 were pooled and freeze-

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dried.

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-

MS analysis

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Prior to MS analysis, purified and dried OS of GM and GP samples were reconstituted

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to a final concentration of 0.1 mg mL-1 with nanopure water, 2’-FL being added as

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internal standard (final concentration of 0.001g L-1). MS analysis was carried out using

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an Agilent 6520 accurate-mass Quadrupole-Time-of-Flight (Q-TOF) LC/MS with a

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microfluidic nano-electrospray chip containing a graphitized carbon enrichment and

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analytical columns (Agilent Technologies, Santa Clara, CA, USA) as previously

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described.

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90% ACN/0.1% formic acid in water (solvent B) at a flow rate of 0.3 µL min-1 for the

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nano pump and an isocratic method (100% A) at a flow rate of 4 µL min-1 for the

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capillary pump were applied. Positive ionization mode with a mass/charge (m/z) range

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of 450–2500 was selected for data acquisition. Automated precursor selection was

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employed based on abundance, with up to 6 MS/MS per MS. A precursor isolation

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window of 1.3 m/z and a fragmentation energy set at 1.8 V/100 Da was used. Reference

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masses of m/z 922.009 and 1221.991 were selected for internal calibration (ESI-TOF

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Low concentration Tuning Mix G1969–85000, Agilent Technologies).

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OS identification was performed using the Find Compounds by Formula function of

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Mass Hunter Qualitative Analysis Version B.06.00 (Agilent Technologies) which

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allowed matching the masses of oligosaccharides with the caprine milk oligosaccharide

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databases, OS composition confirmed by MS/MS as described in literature.

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A binary gradient of 3% ACN/0.1% formic acid in water (solvent A), and

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Abundances of each OS relative to the internal standard (Area OS/Area i.s.) were obtained

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in triplicate.

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Statistical analysis

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Statistica version 7.1 (2005) by Statsoft Inc. (Tulsa, USA) was used for statistical

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analyses. One-way ANOVA test followed by a Fisher test as a post hoc comparison of

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means were used to determine significant differences (P