Using SLIM-Based IMS-IMS Together with Cryogenic Infrared Spectroscopy for Glycan Analysis.

The isomeric heterogeneity of glycans poses a great challenge for their analysis. While combining ion mobility spectrometry (IMS) with tandem mass spectrometry is a powerful means for identifying and characterizing glycans, it has difficulty distinguishing the subtlest differences between isomers. Cryogenic infrared spectroscopy provides an additional dimension for glycan identification that is extremely sensitive to their structure. Our approach to glycan analysis combines ultrahigh-resolution IMS-IMS using structures for lossless ion manipulation (SLIM) with cryogenic infrared spectroscopy. We present here the design of a SLIM board containing a series of on-board traps in which we perform collision-induced dissociation (CID) at pressures in the millibar range. We characterize the on-board CID process by comparing the fragments generated from a pentapeptide to those obtained on a commercial tandem mass spectrometer. We then apply our new technique to study the mobility and vibrational spectra of CID fragments from two human milk oligosaccharides. Comparison of both the fragment drift times and IR spectra with those of suitable reference compounds allows us to identify their specific isomeric form, including the anomericity of the glycosidic linkage, demonstrating the power of this tool for glycan analysis.

Mass Spectrometric Quantification of N-Linked Glycans by Reference to Exogenous Standards.

Environmental and metabolic processes shape the profile of glycoprotein glycans expressed by cells, whether in culture, developing tissues, or mature organisms. Quantitative characterization of glycomic changes associated with these conditions has been achieved historically by reductive coupling of oligosaccharides to various fluorophores following release from glycoprotein and subsequent HPLC or capillary electrophoretic separation. Such labeling-based approaches provide a robust means of quantifying glycan amount based on fluorescence yield. Mass spectrometry, on the other hand, has generally been limited to relative quantification in which the contribution of the signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total profile. Relative quantification has been valuable for highlighting changes in glycan expression between samples; sensitivity is high, and structural information can be derived by fragmentation. We have investigated whether MS-based glycomics is amenable to absolute quantification by referencing signal intensities to well-characterized oligosaccharide standards. We report the qualification of a set of N-linked oligosaccharide standards by NMR, HPLC, and MS. We also demonstrate the dynamic range, sensitivity, and recovery from complex biological matrices for these standards in their permethylated form. Our results indicate that absolute quantification for MS-based glycomic analysis is reproducible and robust utilizing currently available glycan standards.

Evaluation of desialylation during 2-amino benzamide labeling of asparagine-linked oligosaccharides.

Labeling of released asparagine-linked (N-linked) oligosaccharides from glycoproteins is commonly performed to aid in the separation and detection of the oligosaccharide. Of the many available oligosaccharide labels, 2-amino benzamide (2-AB) is a popular choice for providing a fluorescent product. The derivatization conditions can potentially lead to oligosaccharide desialylation. This work evaluated the extent of sialic acid loss during 2-AB labeling of N-linked oligosaccharides released from bovine fetuin, polyclonal human serum immunoglobulin G (IgG), and human α1-acid glycoprotein (AGP) as well as of sialylated oligosaccharide reference standards and found that for more highly sialylated oligosaccharides the loss is greater than the <2% value commonly cited. Manufacturers of glycoprotein biotherapeutics need to produce products with a consistent state of sialylation and, therefore, require an accurate assessment of glycoprotein sialylation.

Review of fiber methods and applicability to fortified foods and supplements: choosing the correct method and interpreting results.

Fiber is known to be an important part of our nutrition and has many positive health benefits, including weight management and maintaining heart health. In recent years, a number of new ingredients have been manufactured or isolated that are being used to increase the health benefits of a product. Some are used as prebiotics that stimulate the growth of the beneficial bacteria in the gut, or are used as replacements for sugars, starch, or fat in manufactured foods. Fiber supplements have also been produced that can be taken to provide additional fiber to the diet. The term "fiber" does not relate to a single analyte or entity, but instead relates to a multitude of components. This adds to the complexity of analytical testing as there are a number of AOAC International and AACC International official methods which have been validated and can be used. Although methods have been developed for specific fiber ingredients, a number of methods have also been developed to capture just "fiber". These "fiber" methods will capture differing degrees of the different fiber ingredients, so knowledge of the fiber sources is critical. The net result is that a variety of testing approaches may be used, but caution must be exercised in order to ensure that the total fiber result is accurately determined. A critical review of available fiber methodology and possible testing approaches is presented, along with how to accurately interpret and understand results.

Industry and regulatory experience of the glycosylation of monoclonal antibodies.

We surveyed 23 antibody-related marketing applications for glycoform analytical and functional information. Our database analysis shows a clear trend of increasing sophistication of analytical methods used to identify and quantify glycans. These have revealed a high degree of complexity and heterogeneity of glycans attached to antibody products. The nature of the complexity is influenced by product type and expression system, and may be associated with functional consequences in some but not all cases.

Assessing the effects of different prebiotic dietary oligosaccharides in sheep milk ice cream.

The objective of this study was to assess the effects of different prebiotic dietary oligosaccharides (inulin, fructo-oligosaccharide, galacto-oligossacaride, short-chain fructo-oligosaccharide, resistant starch, corn dietary oligosaccharide and polydextrose) in non-fat sheep milk ice cream processing through physical parameters, water mobility and thermal analysis. Overall, the fat replacement by dietary prebiotic oligosaccharides significantly decreased the melting time, melting temperature and the fraction and relaxation time for fat and bound water (T22) while increased the white intensity and glass transition temperature. The replacement of sheep milk fat by prebiotics in sheep milk ice cream constitutes an interesting option to enhance nutritional aspects and develop a functional food.

Use of graphitised carbon negative ion LC-MS to analyse enzymatically digested glycosaminoglycans.

Capillary liquid chromatography-mass spectrometry using graphitised carbon stationary phase and ion trap mass spectrometry was shown to be a powerful technique for analysing glycosaminoglycans digested with endoglycosidases. Commonly found disaccharides from heparin/heparan sulphate digests at sub nanomole levels were found to be separated by mass and/or retention time and detected by negative ion electrospray mass spectrometry predominantly as [M-H]- ions using a standard electrospray interface and flow rate between 6-10 microL/min. Graphitised carbon liquid chromatography-fragmentation mass spectrometry provided sequence data of disaccharides and oligosaccharides. Sequence information was obtained from either collision of the [M-H]- ions (low sulphated disaccharides) or of the [M+Na-2H]- ions (highly sulphated disaccharides). This separation and identification method of endoglycosidase digestion and sample preparation using a combination of cation exchange and graphitised carbon, was used to successfully analyse digests of keratan sulphate (keratanase) and heparin (heparinase) standards, and hyaluronic acid (hyaluronidase) from synovial fluid samples.

Growth performance and intestinal microbial populations of growing pigs fed diets containing sucrose thermal oligosaccharide caramel.

Four experiments were conducted to determine growth performance and changes in intestinal microbial populations of growing pigs fed diets containing sucrose thermal oligosaccharide caramel (STOC). Ninety-six barrows and 96 gilts were group-fed experimental nursery diets for 32 d after weaning in both Exp. 1 and 2. For each experiment, pigs were divided into four groups of 48 pigs and were fed either control, antibiotic (Apramycin sulfate, 34 mg/kg), 1% STOC, or 2% STOC diets for 32 d after weaning. Each diet was replicated six times with eight pigs per replication. Pigs were either orally gavaged (Exp 1) with water of STOC (2 g per pig) or pigs were creep-fed (Exp 2) either a control diet or a 2% STOC diet for 5 d before weaning (33 d). At the end of Exp 1 and 2, cecal material was collected for enumeration of total aerobes, total anaerobes, coliforms, lactobacilli, and bifidobacteria. Gilts (96 per experiment) used in Exp. 3 and 4 were weaned at 26 d and fed experimental nursery diets for 32 d. They were fed either a control or 1% STOC diet and were otherwise treated as previously described. There were no significant effects of STOC or antibiotic on ADG, ADFI, feed efficiency, or cecal microbial populations in pigs in this study. Feeding diets containing either antibiotic of STOC did not improve animal performance or change intestinal bacterial populations in the present study.

Analysis of legume oligosaccharides by high-resolution gas chromatography.

The suitability of high-resolution gas chromatography (HGRC) for the analysis of the raffinose family oligosaccharides (raffinose, stachyose, verbascose) was investigated. Aqueous methanol (80%) extracts of pea flour were dried and derivatized with either trimethylimidazole or N-methyl-bis(trifluoroacetamide). Separation of the sugar derivatives was achieved utilizing a 10-m DB5-60W capillary column. The effects of carrier gas (He) flow-rate and split ratio on resolution and reproducibility were studied. HRGC analysis was characterized by excellent resolution and satisfactory reproducibility, and proved to be a rapid, sensitive method for quantitation of oligosaccharides in pea flours.

High-performance capillary electrophoresis of sialylated oligosaccharides of human milk.

Oligosaccharides in human milk inhibit enteric pathogens in vitro and in vivo. Neutral milk oligosaccharides vary among individuals and over the course of lactation. To study such variation in the acidic milk oligosaccharides, a sensitive, convenient, quantitative method is needed. High-performance capillary electrophoresis of underivatized acidic oligosaccharides with detection by UV absorbance at 205 nm proved to be sensitive to the femtomole level. Eleven standard oligosaccharides ranging from tri- to nonasaccharide (3'-sialyllactose, 6'-sialyllactose, 3'-sialyllactosamine, 6'-sialyllactosamine, disialyltetraose, 3'-sialyl-3-fucosyllactose, sialyllacto-N-tetraose-a, sialyllacto-N-tetraose-b, sialyllacto-N-neotetraose-c, disialyllacto-N-tetraose, and disialomonofucosyllacto-N-neohexaose) were resolved; baseline resolutions of 3'-sialyllactose, 6'-sialyllactose, and other structural isomers were achieved. Peak areas were linear from 30 to 2000 pg and were reproducible with a coefficient of variation between 4 and 9%. There was no evidence of quantitative interference of one oligosaccharide with another. In studies using pooled human milk, addition of increasing amounts of authentic standard oligosaccharides produced the expected positive increments in detected values, indicating quantitative recovery without interference by other milk components. The identities of the major sialylated acidic oligosaccharides of pooled human milk agreed with the results of previous studies employing other analytical methods. Comparison of oligosaccharide profiles of milk samples from different donors revealed extensive variation, especially in the structural isomers of sialyllacto-N-tetraose. This sensitive, highly reproducible method requires only simple sample workup and is useful in defining variations in human milk acidic oligosaccharides and investigating their possible relationship with diseases of infants.

Analysis of the neutral glycan fractions of glycosyl-phosphatidylinositols by thin-layer chromatography.

The radiolabeled neutral glycan fractions of both glycosyl-phosphatidylinositol (GPI) protein anchors and related glycolipids were analyzed by thin-layer chromatography on silica gel 60, using butanol:ethanol:water (4:3:3, v/v) or a combination of 1-propanol:acetone:water (9:6:5, v/v and 5:4:1, v/v) as solvents. Dextran acid hydrolysates were used as standards, and oligomers up to 11 glucose units could be resolved. A comparison of 18 GPI-glycan standards revealed that their migration was dependent mainly on the size of the oligosaccharide. Isomers were generally not resolved, with the exception of Man alpha 1-6Man alpha 1-4AHM and Man alpha 1-3Man alpha 1-4AHM. Structures containing galactofuranose or GalNAc were well resolved from structures containing only Galp and/or Manp. The utility of this method for the microsequencing of radiolabeled neutral glycans derived from two GPI glycolipids, using exoglycosidases and chemical treatments, is demonstrated. This method is a simple and useful complement to the existing chromatographic techniques.

A detailed analysis of neutral and acidic carbohydrates in human milk.

Reverse- and normal-phase chromatography have been used to separate a number of standard human milk oligosaccharides derivatized via a reductive amination reaction with 2-aminoacridone (2-AMAC). Analytes were detected by spectrofluorimetry and injected simultaneously with a hydrolyzed dextran ladder derivatized with methyl-4-aminobenzoate. The latter probe does not fluoresce at the wavelength of emission by the 2-AMAC derivatives, and the derivatized, hydrolyzed dextran components were visualized by their ultraviolet absorbance. This procedure gave precise measurements of the "size" of 2-AMAC oligosaccharides in terms of their glucose equivalent values. Analytical amounts of 2-AMAC oligosaccharide standards were also isolated for further characterization by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry. MS-MS was also used to provide information on oligosaccharide sequences. This methodology was used successfully to characterize mixtures of neutral and acidic oligosaccharides from samples of human milk. This approach could be usefully applied to the study of glycoforms from a variety of samples such as those released from glycoproteins/glycolipids; these have been reported to be altered in a number of diseases, for example, cancer, cystic fibrosis, and autoimmune disease such as rheumatoid arthritis.

Glycosylation and biological activity of CAMPATH-1H expressed in different cell lines and grown under different culture conditions.

CAMPATH-1H (where CAMPATH is a trade mark of Wellcome group companies), a humanized IgG antibody used in the therapy of lymphoma, leukaemia and rheumatoid arthritis, has been expressed in Chinese hamster ovary, Y0 myeloma and NS0 myeloma cell lines. These engineered cell lines were grown under different culture conditions, and the antibody isolated and purified. N-Linked oligosaccharides, on the CH2 heavy chain region of the antibody, were isolated and analysed by hydrazinolysis, high-performance anion-exchange chromatography with pulsed amperometric detection, laser-desorption mass spectrometry and sequential exoglycosidase treatment. Both the glycosylation pattern and the biological activity of CAMPATH-1H, as measured by antibody-dependent cell-mediated cytotoxicity, were markedly affected by the cell line used to express the antibody. It is concluded that glycosylation of the antibody may be important in the clinical outcome of therapy.