N-linked Glycan vs PNGase F Released N-glycans from both Human and Bovine Milk - Lab Report Example

An Analysis of N-linked glycan from PNGase F released N-glycans from both human and bovine milk Name Institution Instructor Course Date Introduction Human milk contains oligosaccharides (glycans) which play a very imperative role as selective growth substrates for some microbial flora in the human gastrointestinal system. Bovine milk is amongst the many commercially available sources of these unique molecules found in human milk. It contains some proteins attached to glycans designed to mimic the human proteins. Researches have shown that these kinds of reactions between the glycans and protein have a potential of affecting the product in a number of ways. For instance, it could affect the thermal stability of the product, its physiochemical properties immunogenicity and its half-life (Li and d'Anjou 2009). It has also been said that bovine milk contains different structures and concentrations from those of human milk. In order to ensure safe usage of glycoprotein products, as a biopharmaceutical intervention, it is imperative to ascertain the heterogeneous structure of carbohydrate moieties attached to the glycoprotein. This is also important for the manufacturers of the products during the manufacturing process in order to ensure production of quality products and for marketing purposes as they write the nutritional value of the products in the packaged milk (Demain and Vaishnav 2009). This is a report which was the results of a lab practical, ran for two weeks by the author of this report and a class mate. The practical was aimed at analyzing and comparing the N-glycan compositions of two spectra of PNGase F released N-glycans from both human and bovine milk samples. The release glycans were then analyzed using liquid chromatography (LC) coupled with Electrospray Ionization (ESI) mass spectrometer, after a chromatographic separation using a graphitized carbon chip which was porous (Zaia 2010). Materials The following materials were used during the practical: 50µg lactoferrin purified from human and bovine milk samples (from a 1 mg/mL solution) ; 0.5M DTT (Dithiothreitol); 1M Iodoacetamide Made Freshly ; 2 TopTip EMPTY pipette tips from Glygen corp PNGase F (2 x 0.5 Units from a 0.5 Unit/µL solution) For the class; 100 % Methanol Glacial acetic acid 1 M sodium borohydride (NaBH4 ) in 50mM Potassium hydroxide (KOH) ; Freshly Made Cation exchange resin- Dowex AG 50W X8 1 M; Hydrocholoric acid ; 100 mM Ammonium acetate (CH3COON H4), pH 5 Eppendorf tubes (1.5ml); Glass Autosampler Vials (Waters) and 50°C waterbath 37°C incubator Refrigerated vacuum concentrator Methods Preparation of the samples During the first week, we prepared the samples. We got a sample of human milk lactoferrin (catalogue no. L0520, Sigma-Aldrich, Australia) and one sample of bovine lactoferrin (catalogue no. L9507, Sigma- Aldrich, Australia). 50µg of glycoprotein in 50µL of water. Reduction and alkylation of glycoproteins and Reduction of the N-linked glycans The initial step involved reduction of the disulphide bonds in the samples, in order to unfold the proteins in them (Demain & Vaishnav, 2009). This was done using 10mM DTT under a temperature of 56°C for 30minutes. After this we prepared 1M Iodoacetamide (MW: 185 g/mol). We then alkylated the proteins in order to prevent the free cysteine from refolding the glycoprotein. This was done using 20mM Iodoacetamide (1µL of 1M Iodoacetamide per sample). The environment was maintained dark and the process ran for duration of 30 minutes. The final step during this week involved the release of -linked glycans from lactoferrin using PNGase F. 1µL of PNGase F (0.5 Units/µL) was added to each sample and they were incubated overnight at a temperature of 37˚C. The samples were then stored at a temperature of 20˚C in a freezer, the next day. During the second week of the practical the following were done: Reduction of the N-linked glycans 10 µL of 100 mM ammonium acetate pH 5 was added to the samples and they were incubated at room temperature for duration of 10 minutes. This was aimed at removing amines from the glycans to ease the process of their reduction. The solution was then evaporated to dryness in a vacuum concentrator. 20µL 1M NaBH4 in 50mM KOH and incubate for 1h at 50˚C were added and incubated for a duration of one hour at a temperature of 50˚C. This was aimed at reducing the released glycans to non-anomeric oligosaccharide alditols. The samples were then acidified by adding 2 µL glacial acetic acid and mixing vigorously. During this process, Effervescence was observed which could have been related to release of H2 (Demain & Vaishnav, 2009). The solutions were spanned briefly then they were desalted using the cation exchange columns. The cation exchange micro columns were prepared using the following procedure: cation exchange resin (Dowex AG 50W X8) was deposited into a TipTop EMPTY tip to a final packed bed volume of 30 µL. the pack was then washed with 2 x 60 µL 1M HCl (to remove already bound contaminating salts); 2 x 60 µL methanol (to remove already bound organic contaminants); (this was done by positioning the micro columns in 1.5 mL Eppendorf tubes and using a small table spinner/centrifuge for spinning through each wash). The cation exchange micro column was transferred into a new Eppendorf tube, and 2 x 50 µL water was added to the top of the packing, then it was span through. A little water was left on top and the tube lid was closed, making it ready for storage of the columns. Desalting of the reduced N-linked glycans The micro columns were placed in new Eppendorf tubes and the samples were applied on top of the packing then the sample was spin into themicro column.The original Eppendorf sample tube with 10 µL water and apply to the top of the micro column. The original Eppendorf sample tube was washed out with 10 µL water and applied to the top of the micro column. This was then spinning through into fresh Eppendorf tube. 25 µL of water was added to the micro column in order to elute the glycan. It was spinning again through into the same Eppendorf tube. The micro columns were removed and the eluted glycans was dried in the Eppendorf tubes in the vacuum concentrator. 50 µL methanol was added to the samples and then the samples were driedin the SpeedVac concentrator. This procedure was repeated one more time. This was to remove residual borate as the volatile methyl borate. A lot of care was taken during this procedure in order to ensure that the dried samples were completely re-dissolved in methanol before each re-drying step. The N-glycans were then taken up in 50 µL water and pipetted into the glass auto sampler vials provided for analysis on the Liquid Chromatography Mass Spectrometer. The glass vials containing the sample were queued in the HPLC auto samplerwhich was connected to the mass spectrometer. The samples were subjected to separation of the oligosaccharides on a capillary LC-ESI- MS in the negative ion mode using a Agilent 6330 ion-trap mass spectrometer. The samples were applied to a HyperCarb porous graphitized carbon HPLC column (5 μm Hypercarb, 0.32 × 150 mm, Thermo Hypersil, Runcorn UK). The N-linked glycans released from lactoferrin by PNGase F were separated using a linear gradient of 0-45% (v/v) Acetonitrile/10 mM NH4HCO3 for 85 min, at a flow rate of 2 μL/min. Table 1 A summed mass spectrum showing the mass of each released N-glycan Monosaccharaides Residual mass (Da) Galactose (Gal) 162 Glucose (Glc) 162 Mannose (Man) 162 Fucose (Fuc) 146 Xylose (Xyl) 132 N-acetylglucosamine (GlcNAc) 203 N-acetylgalactosamine (GalNAc) 203 N-acetyl neuraminic acid (sialic acid) 291 N-glycolyl neuraminic acid 306 Results The results revealed a complex glycan pool, which comprised of complex sialylated fucosylated glycans, which were mainly diantennary, triantennary and tetraantennary. The pool also comprised of high mannose, some hybrid and some common core str. Subunits. The human milk N-glycan (figure 1A) resembles 38 N-glycan compositions. On the other hand, the bovine milk (figure 1B) the comprised of 51 N-glycans. Figure 2.My results  The results from the two chromatogram (figure 2), were reflecting that the elution pattern was mainly starting with high mannose, which was followed by neutral complex glycans then sialylated glycans. The findings were similar to those presented by the lecturer.Additionally; it was observed that there was high mannose sialylation and fucosylation, in both human and bovine milk. It was also observed that 25 out of the 38 human N-glycan compositions were fucosylated. On the other hand, only 21 out of the 51 N-glycans in bovine milk were fucosylated. Additionally, approximately 60% of the fucosylated human glycan were visible with higher degrees of fucosylation (di-, tri- and tetra-). On the other hand, only 30% of the fucosylated bovine glycan could be observed using higher degrees of fucosylation. With reference to sialylation 12 out of the total 38 glycans in human milk were seen to be sialylated as compared to 22 out of 51 glycans of bovine milk. Finally, while only a tenth of all the silyated human milk glycan had a multiple sialyation, 30% of bovine milk glycans had a multiple sialyation (table 2). Table 2 comparison of features between human and bovine milk The LC /ESI analyses enabled us to quantify the different N-glycan types found in both human and bovine milk. The pie charts in figure 4 shows the abundances of the different N-glycan types. They also show the fucosylation distribution in both human and bovine milk. From the findings, 75% of the totals N-glycan are fucosylated in the human milk, while only 31% of the totals are fucosylated in the bovine milk. Figure 4 Pie charts showing the relative abundances of (A) all the N-glycan types in human milk; (B) Fucosylated N-glycans in human milk. (C) All the N-glycan types in bovine milk. (D) Fucosylated N-glycans in bovine milk Discussion The observed number of glycan composition in both bovine and human milk is equivalent to more than a hundred compounds when putting into consideration isomers. However, they also reflect that the number of glycans is not indefinitely large, hence displaying simplicity to some extent.  The results from the two chromatogram, (figure 1) were reflecting that the elution pattern was mainly starting with high mannose, which was followed by neutral complex glycans then sialylated glycans. The findings were similar to those presented by the lecturer, meaning that sialylated glycans pocess a higher affinity to graphitized carbon as compared to neutral glycans. High mannose glycan have an earlier procession time as compared to low mannose glycans (Demain & Vaishnav, 2009).  The method used enabled us to establish some distinguishing features between the human and bovine milk. For instance, In terms of glycan composition we were able to observe high mannose sialylation and fucosylation, which were observed in both chromatograms (figure 1). In addition, despite all the five high mannose glycan being present in both the milk and bovine chromatograms, there were some relevant differences which were observed with reference to fucosylated and sialylated glycan composition. To begin with, it was discovered that 25 out of the 38 human N-glycan compositions were fucosylated. On the other hand, only 21 out of the 51 N-glycans in bovine milk were fucosylated. Additionally, approximately 60% of the fucosylated human glycan were visible with higher degrees of fucosylation (di-, tri- and tetra-). On the other hand, only 30% of the fucosylated bovine glycan could be observed using higher degrees of fucosylation. With reference to sialylation 12 out of the total 38 glycans in human milk were seen to be sialylated as compared to 22 out of 51 glycans of bovine milk. Finally, while only a tenth of all the silyated human milk glycan had a multiple sialyation, 30% of bovine milk glycans had a multiple sialyation (table 2). This suggests that bovine milk has a less fucosylation and a higher sialylation as compared to human milk. The results suggest that while high fucosylation and sialylation are general features of the human milk N-glycome, bovine milk is significantly less fucosylated and more highly sialylated Table 2. Results on the general features in glycosylation in human and bovine milk An additional difference between glycans from human and bovine milk is that bovine glycans contain a NeuGc residues, which is absent in human milk glycan. In addition, 5% of sialylation abundance found in the bovine milk, was in correspondence to NeuGc residue. However both samples contained NeuAc. According to the results from this practical, a significant difference was observed in terms of sialytaion between human and bovine milk. Similar studies had been carried out before and reflected the same. This is suggestive that protein bound glycans are a potential source of sialylation in infants who breastfeed on their mother’s milk. In this study, 75% of human milk was observed to be fucosylated. This was closely similar to studies carried out before, which reflected 70% fucosylation. The results are suggestive of the point that in bovine milk, protein bound N-glycan are a main source of fucosylation (Demain & Vaishnav, 2009). Conclusion The practical used LC-ESI approach to analyze N-glycans in both human and bovine milk. The N-Glycans were separated using carbon chip depending on their unique types and their monosaccharide composition. 38 N-glycan composition was observed in human milk ad 51 in bovine milk. The human milk was observed to contain only NeuAc while the Bovine milk was seen to contain an additional NeuGc. The main aim of the practical; which was to analyse the N-glycan composition of the bovine and human milk were met. 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