Characterization of recombinant human lactoferrin N-glycans expressed in the milk of transgenic cows.

Lactoferrin (LF) is one of the most abundant bioactive glycoproteins in human milk. Glycans attached through N-glycosidic bonds may contribute to Lactoferrin functional activities. In contrast, LF is present in trace amounts in bovine milk. Efforts to increase LF concentration in bovine milk led to alternative approaches using transgenic cows to express human lactoferrin (hLF). This study investigated and compared N-glycans in recombinant human lactoferrin (rhLF), bovine lactoferrin (bLF) and human lactoferrin by Nano-LC-Chip-Q-TOF Mass Spectrometry. The results revealed a high diversity of N-glycan structures, including fucosylated and sialylated complex glycans that may contribute additional bioactivities. rhLF, bLF and hLF had 23, 27 and 18 N-glycans respectively with 8 N-glycan in common overall. rhLF shared 16 N-glycan with bLF and 9 N-glycan with hLF while bLF shared 10 N-glycan with hLF. Based on the relative abundances of N-glycan types, rhLF and hLF appeared to contain mostly neutral complex/hybrid N-glycans (81% and 52% of the total respectively) whereas bLF was characterized by high mannose glycans (65%). Interestingly, the majority of hLF N-glycans were fucosylated (88%), whereas bLF and rhLF had only 9% and 20% fucosylation, respectively. Overall, this study suggests that rhLF N-glycans share more similarities to bLF than hLF

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

A detailed qualitative and quantitative characterization of goat colostrum oligosaccharides (GCO) has been carried out for the first time. Defatted and deproteinized colostrum samples, previously treated by size exclusion chromatography (SEC) to remove lactose, were analyzed by nanoflow liquid chromatography–quadrupole-time of flight mass spectrometry (Nano-LC-Chip–Q-TOF MS). Up to 78 oligosaccharides containing hexose, hexosamine, fucose, N-acetylneuraminic acid or Nglycolylneuraminic acid monomeric units were identified in the samples, some of them detected for the first time in goat colostra. As a second step, a hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS) methodology was developed for the separation and quantitation of the main GCO, both acidic and neutral carbohydrates. Among other experimental chromatographic conditions, mobile phase additives and column temperature were evaluated in terms of retention time, resolution, peak width and symmetry of target carbohydrates. Narrow peaks (wh: 0.2–0.6 min) and good symmetry (As: 0.8–1.4) were obtained for GCO using an acetonitrile:water gradient with 0.1% ammonium hydroxide at 40 ◦C. These conditions were selected to quantify the main oligosaccharides in goat colostrum samples. Values ranging from 140 to 315 mg L−1 for neutral oligosaccharides and from 83 to 251 mg L−1 for acidic oligosaccharides were found. The combination of both techniques resulted to be useful to achieve a comprehensive characterization of GCO.This work has been funded by Junta de Andalucía (project AGR2011-7626), CSIC (project i-link0827), Comunidad Autónoma de Madrid (Spain) and European funding from FEDER program (AVANSECAL-CM S2013/ABI-3028) and Fundación Ramón Areces. This work was also supported by the UC Davis RISE program and the National Institutes of Health awards R21AT006180, R01AT007079, R01AT008759-02.Peer reviewe

Characterization of recombinant human lactoferrin N-glycans expressed in the milk of transgenic cows

Lactoferrin (LF) is one of the most abundant bioactive glycoproteins in human milk. Glycans attached through N-glycosidic bonds may contribute to Lactoferrin functional activities. In contrast, LF is present in trace amounts in bovine milk. Efforts to increase LF concentration in bovine milk led to alternative approaches using transgenic cows to express human lactoferrin (hLF). This study investigated and compared N-glycans in recombinant human lactoferrin (rhLF), bovine lactoferrin (bLF) and human lactoferrin by Nano-LC-Chip-Q-TOF Mass Spectrometry. The results revealed a high diversity of N-glycan structures, including fucosylated and sialylated complex glycans that may contribute additional bioactivities. rhLF, bLF and hLF had 23, 27 and 18 N-glycans respectively with 8 N-glycan in common overall. rhLF shared 16 N-glycan with bLF and 9 N-glycan with hLF while bLF shared 10 N-glycan with hLF. Based on the relative abundances of N-glycan types, rhLF and hLF appeared to contain mostly neutral complex/hybrid N-glycans (81% and 52% of the total respectively) whereas bLF was characterized by high mannose glycans (65%). Interestingly, the majority of hLF N-glycans were fucosylated (88%), whereas bLF and rhLF had only 9% and 20% fucosylation, respectively. Overall, this study suggests that rhLF N-glycans share more similarities to bLF than hLF

Comparison of bLF, rhLF and hLF <i>N-</i>glycan compositions.

<p>Comparison of bLF, rhLF and hLF <i>N-</i>glycan compositions.</p

PCA plot of composition of released <i>N-</i>glycans of human lactoferrin (hLF), recombinant human lactoferrin (rhLfa) and bovine lactoferrin (bLf).

<p>Of the variance, 62.45% was explained by the first principal component and 36.97% was explained by the second principal component.</p

Deconvoluted tandem spectrum of the neutral N-glycan 5Hex2HexNAc from recombinant human lactoferrin.

<p>This glycan corresponds to 1235.44 m/z. Green circles and blue squares represent mannose and HexNAc residues, respectively.</p

Heatmap of compound abundances associated with lactoferrin from different sources.

<p>Compound relative abundances were standardized (Z score, shown in legend) prior to unsupervised hierarchical clustering of samples (rows). Compound identity is noted below each column.</p

High mannose, sialylated and neutral complex/hybrid <i>N</i>-glycans released from hLF, rhLF and bLF by PNGase F.

<p>Tukey’s test was used to indicate significant differences (p<0.05) between groups. Same letters indicate no significant difference.</p

Comprenhesive characterization of goat colostrum oligosacharides by liquid chomatography coupled to mass spectrometry

Resumen del póster presentado a la XV Reunión Científica de la Sociedad Española de Cromatografía y Técnicas Afines (SECyTA) y la VII Reunión Nacional de la Sociedad Española de Espectrometría de Masas (SEEM); celebradas en Castellón del 27 al 30 de octubre de 2015.Goat milk is a complex mixture of nutritive and bioactive components with reported health benefits such as carbo-hydrates, lipids and proteins. Although lactose is the main carbohydrate, presence of other oligosaccharides (OS) similar to those found in human milk, has been reported. Some of these oligosaccharides, such as those containing fucosyl- or sialyl- groups have been described to have prebiotic and pathogen binding activities. In recent years, different analytical techniques have demonstrated their potential in OS characterization. Nanoflow Liquid Chromatography-Quadrupole-TOF MS (Nano-LC-Chip-Q-TOF MS) shows a high sensitivity and capacity for OS compositional verification, whereas, Hydrophilic Interaction Liquid Chromatography (HILIC) is a powerful LC operation mode for quantitative analysis of OS, providing appropriate resolution and good peak shapes. In this work, qualitative and quantitative analysis of goat colostrum oligosaccharides (GCO) has been carried out. Milk colostra, previously defatted and deproteinized, were treated by Size Exclusion Chromatography (SEC) to remove lactose. Up to 78 oligosaccharides containing hexose, hexosamine, fucose, N-acetylneuraminic acid or N-glycolylneuraminic acid monomeric units were identified in the samples by Nano-LC-Chip-Q-TOF MS, some of them detected for the first time in goat colostra. As a second step, a previously optimized HILIC-MS method was used for the separation and quantitation of the main GCO, both acidic and neutral compounds. Values ranging from 140 to 315 mg L-1 for neutral oligosaccharides and from 83 to 251 mg L-1 for acidic oligosaccharides were found. The most abundant OS were galactosyl-lactoses (124.92-265.77 mg L-1), followed by 6’-sialyl-lactoseand sialyl-lactosamine. To the best of our knowledge, this is the first time that a comprehensive characterization of GCO is carried out.Peer reviewe

Recombinant human lactoferrin purification.

<p>(A) Skim milk protein profile analyzed by 12% SDS-PAGE. (B) Elution of rhLF purification by heparin Sepharose. (C) Elution of rhLF purification by ion-exchange chromatography.</p