high-pH Anion-exchange Chromatography With Pulsed Amperometric Detection Research Articles

Urinary excretion of lactose and oligosaccharides in preterm infants fed human milk or infant formula.

At present, not much is known about the absorption and metabolism of human milk (HM) oligosaccharides in term and preterm infants. We investigated the renal excretion of lactose and complex oligosaccharides in preterm infants fed HM (n = 9, mean actual body weight 2290 g) or a cow's milk-based infant formula (n = 9, mean actual body weight 2470 g). We found that the renal excretion of lactose in HM-fed infants was slightly lower than in formula-fed infants (14.0 +/- 7.4 versus 20.4 +/- 8.7 mg kg-1 day-1, mean +/- SD). The excretion of neutral sugars deriving from oligosaccharides was similar in HM-fed and formula-fed infants (3.8 +/- 2.1 versus 2.9 +/- 0.9 mg kg-1 day-1); the difference between means was not statistically significant. The separation and characterization of oligosaccharides by high-pH anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) and subsequent analysis by fast atom bombardment-mass spectrometry (FAB-MS) revealed a more complex pattern in HM-fed infants compared to the formula-fed group. Lactose-derived oligosaccharides characteristic for HM (e.g. lacto-N-tetraose, and lacto-N-fucopentaoses I and II) were excreted in HM-fed but not in formula-fed infants. These results indicate that nutrition has a significant impact on the oligosaccharide composition in urine of preterm infants.

Separation of Human Serum Transferrin Isoforms by High-Performance Pellicular Anion-Exchange Chromatography

Glycoproteins are microheterogeneous with respect to their attached oligosaccharides. When these oligosaccharides contain sialic acid, the oligosaccharide microheterogeneity will impart charge heterogeneity to the glycoprotein. We found that two commercial preparations of human serum transferrin (HST), a sialylated glycoprotein, have very different chromatographic profiles when the samples are separated by pellicular anion-exchange chromatography. Each anion-exchange profile contained multiple peaks, which suggested that both glycoproteins have charge heterogeneity. High-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD) analysis of sialic acids and oligosaccharides released from the two preparations of HST revealed that the two preparations differed in sialic acid and sialylated oligosaccharide content. When oligosaccharides were released from the anion-exchange fractions of the two HST preparations, the HPAEC/PAD oligosaccharide profiles showed that protein retention was directly related to sialylated oligosaccharide content (i.e., the longer a fraction was retained, the greater its sialylated oligosaccharide content). Therefore, the anion-exchange profiles of the two HST preparations are related to their sialylated oligosaccharide content. We believe that pellicular anion-exchange chromatography can be used to quickly monitor gross changes in the sialylation of sialylated glycoproteins due to physiological state, or in the case of recombinant glycoproteins, culture conditions and/or purification.

6] Identification and characterization of glycopeptides in tryptic maps by high-pH anion-exchange chromatography

This chapter describes the application of high-pH anion-exchange chromatography, with pulsed amperometric detection (HPAEC-PAD) to the identification and partial characterization of glycopeptides, within a reversed-phase high-performance liquid chromatography (HPLC) peptide map. HPAEC is a versatile chromatographic method that can be used for the detection and quantitation of monosaccharides, released by acid hydrolysis and for the analysis of intact oligosaccharides released enzymatically or chemically. A large number of other analytical methods, including mass spectrometry (MS), enzymatic microsequencing, nuclear magnetic resonance (NMR), and capillary electrophoresis, can also be applied to the characterization of glycopeptides; in general, a combination of several complementary analytical methods are needed to achieve a definitive carbohydrate structure determination. This chapter discusses the type of information that can be obtained, using a single, comparatively simple, analytical method.

Chemical modification and fractionation of pea stem polysaccharides

Pea stem tissue was fractionated by chemical and enzymatic methods and the water soluble fractions analyzed by NMR spectroscopy and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The obtained results indicate that arabinogalactoxyloglucan in similar purity could be isolated by one-step extraction with 2% NaOH or 5% ZnCl 2, as well as by two-step treatment with pronase and α-amylase. By quaternization under alkaline conditions with 3-chloro-2-hydroxyprophyltrimethylammonium chloride (CHMAC), a mixture of arabinogalactan and galacturonan modified with trimethylammoniumhydroxypropyl (TMAHP) group (10.8% yield) could be isolated. By extraction with 2% NaOH, a fraction in 19.0% yield could be obtained, contrary to 2.4% yield of polysaccharide when treated with 5% ZnCl 2. Treatment with pronase and amylase have given 1.5 or 4.5% yields of soluble frations, while the subsequent residue extracted with ammonium oxalate gave galacturonic acid oligomers (2.1% yield). Direct extraction with (NH 4) 2(COO) 2 and without enzymatic pretreatment solubilized only a small part of the material (0.9% yield) consisting of three polysaccharide fractions having the highest molecular weight from all obtained fractions.

Sialyl-Tn-KLH, glycoconjugate analysis and stability by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

The quantitation of sialyl-Tn (STn) conjugated to keyhole limpet haemocyanin (KLH) can be determined by quantitating the amount of N-acetylneuraminic acid (NANA) released by acid or enzymatic digestion. An optimal 0.1 N H2SO4 acid hydrolysis at 80 degrees C results in quantitative release of NANA with minimal loss. A rapid isocratic method for the quantitation and separation of NANA is described using high-pH anion-exchange chromatography and pulsed amperometric detection (PAD). Multiple injection of NANA standard and/or samples containing protein led to a decrease in the PAD response which was corrected by addition of internal standard, alpha-2-keto-3-deoxyoctonate (KDO). The ratio of NANA/KDO peak area or peak height gives a linear response with increasing amount of NANA in the range 2.5-20 micro g/ml (r2 = 0.99). The limit of quantitation (LOQ) for NANA using this isocratic method is 1.9 micro g/ml (approximately 160 pmol/25 micro l injection). Based on the multiple determination the glycoconjugate, STn-KLH, showed a NANA content of 2.9% (w/w). Acid hydrolysis and the sialidase treatment of STn-KLH both yielded a similar NANA content. The carrier protein, KLH, showed the absence of NANA. The stability of glycoconjugate STn-KLH was monitored by a gradient method which separated possible degradation products STn-crotyl, NANA and GalNAc. Subjecting the glycoconjugate STn-KLH to various stress conditions of temperature, pH and oxidation does not result in any release of sialic acid, GalNac and STn-crotyl group.

The critical glycosylation site of human transferrin receptor contains a high-mannose oligosaccharide.

The human transferrin receptor (TfR) contains three N-linked oligosaccharides and glycosylation is required for the proper folding and function of the molecule. Earlier studies demonstrated that the oligosaccharide at Asn-727 is vital for the production of fully active TfR. The oligosaccharide(s) present at this site have been analysed using a combination of site-directed mutagenesis and chemical analysis. Wild-type TfR and mutants containing only the Asn-727 site or missing all three sites were transfected into mouse 3T3 cells and receptors were analysed by endo-N-acetylglucosaminidase H (Endo-H) digestion, SDS-PAGE and immunoblotting. These studies suggested that the Asn-727 site contains high-mannose or Endo-H-sensitive hybrid oligosaccharides. Glycosylation of Asn-727 found in the TfR purified from human placentae was analysed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and mass spectrometry following tryptic digestion, peptide purification via reverse-phase high-performance liquid chromatography (RP-HPLC) and peptide sequencing. HPAE-PAD showed the presence of a series of high-mannose oligosaccharides. Mass spectrometry confirmed these observations, but also showed the presence of an 80 Da anionic moiety on a fraction of the oligosaccharides.

Structural Characterization of the N-Glycans of a Humanized Anti-CD18 Murine Immunoglobulin G

This study characterized the N-glycans of a humanized immunoglobulin G4 (IgG4) expressed in NS/O mouse myeloma cells and directed against the CD18 family of adhesion-promoting receptors on leukocytes. The N-glycans were released from the purified recombinant IgG by N-glycanase treatment, purified by Sephadex G-50 chromatography, and fractionated by Bio-Gel P-4 chromatography into three oligosaccharide pools. Each pool was analyzed individually by glycosyl composition analysis, high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), 600-MHz 1H-NMR spectroscopy, and electrospray-ionization mass spectrometry. In addition, each of the three pools was subfractionated by HPAEC and the isolated subfractions that contained sufficient material were hydrolyzed and analyzed for glycosyl composition by HPAEC-PAD. The overall results indicate the presence of five oligomannoside-type structures (containing 5 to 8 Man residues) which are not usually found in IgG, and the presence of eight diantennary (mostly truncated) N-acetyllactosamine-type structures which are typical of mouse and human IgGs. The N-acetyllactosamine-type structures were heterogeneous with regard to α(1 → 6) fucosylation of the linkage GlcNAc, and the presence or absence of GlcNAc and/or Gal β(1 → 4)GlcNAc extending the core pentasaccharide (Man3GlcNAc2). No evidence was found for the presence of sialic acid or bisecting GlcNAc residues on the N-acetyllactosamine-type chains. The latter finding suggests that the N-glycans of this humanized IgG are of the mouse type.

Identification, Quantification, and Characterization of Glycopeptides in Reversed-Phase HPLC Separations of Glycoprotein Proteolytic Digests

Monosaccharide analysis by high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD) was used to identify the glycopeptides in the reversed-phase (RP-HPLC) separation of a bovine fetuin tryptic digest (1.6 nmol). This method, requiring no sample derivatization, identified four asparagine-linked (N-linked) glycopeptides and at least seven serine/threonine-linked (O-linked) glycopeptides. Glycopeptide identification was confirmed by Edman sequencing. Monosaccharide quantification of each glycopeptide suggested that all of the N-linked glycopeptides were the complex type and all the O-linked glycopeptides were sialylated. We determined that glycopeptides could be prepared by acidic reversed-phase chromatography with less than 3% loss of N-acetylneuraminic acid (Neu5Ac). The N-linked glycopeptides of bovine fetuin were prepared, digested with N-glycosidase F (PNGase F), and their oligosaccharides analyzed by HPAEC/PAD. These oligosaccharide profiles revealed that the Asn-138 oligosaccharide attachment site contained the majority of the disialylated and monosialylated oligosaccharides. The Asn-158 oligosaccharide attachment site contained the majority of the tetrasialylated oligosaccharides.

The mapping by high-pH anion-exchange chromatography with pulsed amperometric detection and capillary electrophoresis of the carbohydrate moieties of human plasma α1-acid glycoprotein

The reducing oligosaccharides released from α 1-acid glycoprotein (AGP) by conventional hydrazinolysis have been analyzed by two different mapping techniques, using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and capillary electrophoresis (CE) with uv detection at 190 nm. The CE measurements proved about 4000 times more sensitive than the measurements by HPAE-PAD. The N-glycan pool was fractionated by Mono Q anion-exchange chromatography, and individual fractions so obtained were desialylated using Vibrio cholerae neuraminidase. The resulting asialo- N-glycans were further analyzed by HPAE-PAD, revealing 2 major, 4 intermediate, and 4 small peaks and at least 3 spikes, which counted for at least 13 different asialo- N-glycans. The carbohydrate structures were tentatively assigned by comparison of the Mono Q-separated N-glycans with the known AGP carbohydrate structures and known structures contained in a mapping database that allows structural assignment of N-glycans by mere comparison of retention times. In addition to the hitherto known AGP carbohydrate structures, we have tentatively identified a number of sulfated N-glycans that are currently being analyzed in more detail. We have also compared the glycan pools recovered from AGP using hydrazinolysis and glycopeptidase F (PNGase F). Approximately 40 distinct peaks could be detected in the hydrazinolysis-derived N-glycan pool by either technique (HPAE-PAD and CE), while about 30 distinct peaks were detected in the N-glycan pool derived by PNGase F digestion of the tryptic AGP digest of the same batch of AGP. These differences were attributed to an increased desialylation (∼3 mol%) during hydrazinolysis, based on the detection by HPAE-PAD and CE of free sialic acid and monosialylated oligosaccharides in the glycan pool derived by conventional hydrazinolysis. The integrity of the N-glycans' chitobiose core was examined by 500-MHz 1H NMR spectoscopy. The hydrazinolysis procedure could be optimized such that the hydrazinolysis-derived N-glycan pool was chromatographically essentially identical to the PNGase F-derived N-glycan pool. Hydrazinolysis proved best, with practically no loss of N-acetlylneuraminic acid and the closest resemblance to the PNGase F-derived N-glycan pool, using an automated apparatus. Notably, it was recognized that, in our hands, PNGase F digestion in the presence of sodium dodecyl sulfate resulted in partial desialylation of the liberated N-glycans.

A strategy for the mapping of N-glycans by high-pH anion-exchange chromatography with pulsed amperometric detection

We have evaluated the high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with respect to its suitability to establish a carbohydrate mapping database that would enable carbohydrate structural analysis by mere comparison of retention times. The suitability of HPAE-PAD for carbohydrate structural analysis was ascertained by validation experiments. The retention times of distinct N-glycans, prepared and measured on different days, were shown to be highly reproducible, with a coefficient of variation (CV) of <0.5%, requiring less than 100 pmol of N-glycan per injection for reliable measurements. Including appropriate internal chromatographic standards, such as (Neu5Ac)1, (Neu5Ac)2, (Neu5Ac)3, and Neu5Gc, the HPAE-PAD method fulfills the analytical requirements with respect to accuracy, precision, reproducibility, and sensitivity. The N-glycan mapping database was established, using two optimized linear gradients “S” and “A” for sialylated and asialo N-glycans, respectively. Approximately 100 different N-glycans of known structure, which have thus far been measured and characterized, have entered our Lotus 1-2-3 mapping database. The efficiency of the database for structural determinations was tested, using the N-linked carbohydrates isolated from rhuEPO, expressed in BHK cells. Nine different sialylated N-glycans of rhuEPO (BHK) could be assigned with a deviation of <±0.5%, using gradient S, and six of the eight asialo N-glycans of rhuEPO (BHK) detected with gradient A could be assigned with an accuracy of <±1%, three of them even with an accuracy of <0.1%, proving the reliability of the established HPAE-PAD mapping database.