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Conventional mass spectrometry (MS)-based analytical methods for small carbohydrate fragments (oligosaccharides, degree of polymerization 2–12) are time-consuming due to the need for offline sample pretreatment such as desalting. Herein, we report a new paper spray ionization method, named desalting paper spray (DPS), which employs a piece of triangular filter paper for both sample desalting and ionization. Unlike regular paper spray ionization (PSI) and nano electrospray ionization (nanoESI), DPS-MS allows fast and sensitive detection of oligosaccharides in biological samples having complex matrices (e.g., Tris, PBS, HEPES buffers or urine). When oligosaccharide sample is loaded onto the filter paper substrate (10 mm × 5 mm, height × base) made mostly of cellulose, oligosaccharides adsorb on the paper via hydrophilic interactions between oligosaccharides and cellulose. Salts and buffers can be washed away using an ACN/H(2)O (90/10 v/v) solution, While oligosaccharides can be eluted from the paper using a solution of ACN/H(2)O/formic acid (FA) (10/90/1 v/v/v) and directly spray-ionized from the tip of the paper. Various saccharides at trace levels (e.g., 50 fmol) in nonvolatile buffer can be quickly analyzed by DPS-MS (< 5 min per sample). DPS-MS is also applicable for direct detection of oligosaccharides from glycosyltransferase (GT) reactions, a challenging task which typically requires radioactive assay. Quantitative analysis of acceptor and product oligosaccharides shows increased product with increased GT enzymes used for the reaction, a result in line with radioactivity assay. This work suggests that DPS-MS has potential for rapid oligosaccharides analysis from biological samples.

Paper-based cultures are an emerging platform for preparing 3D tissue-like structures. Chemical gradients can be imposed upon these cultures, generating microenvironments similar to those found in poorly vascularized tumors. There is increasing evidence that the tumor microenvironment is responsible for promoting drug resistance and increased invasiveness. Acidosis, or the acidification of the extracellular space, is particularly important in promoting these aggressive cancer phenotypes. To better understand how cells respond to acidosis there is a need for 3D culture platforms that not only model relevant disease states but also contain sensors capable of quantifying small molecules in the extracellular environment. In this work, we describe pH-sensing optodes that are capable of generating high spatial and temporal resolution maps of pH gradients in paper-based cultures. This sensor was fabricated by suspending microparticles containing pH-sensitive (fluorescein) and pH-insensitive (diphenylanthracene) dyes in a polyurethane hydrogel, which was then coated onto a transparent film. The pH-sensing films have a fast response time, are reversible, stable in long-term culture environments, have minimal photobleaching, and are not cytotoxic. These films have a pKa of 7.61 ± 0.04 and are sensitive in the pH range corresponding to normal and tumorigenic tissues. With these optodes, we measured the spatiotemporal evolution of pH gradients in paper-based tumor models.

Krill are filter feeders that consume algae, plankton and detritus, indicating that krill possess an adequate cellulose digesting system. However, less is known about the enzymatic properties of crustacean cellulases compared to termite cellulases. In the present study, 48 kDa-cellulase was highly purified from krill ( Euphausia pacifica ) in an effort to determine the cleavage specificity of the enzyme. The most notable characteristic of the enzyme was its high activity against both lichenan and carboxymethyl cellulose. The enzyme hydrolyzed internal β-1,4 glycosidic bonds within lichenan as well as carboxymethyl cellulose to release oligosaccharides and glucose. The effects of pH and temperature on the activity and stability of both enzyme activities were almost identical. Cello-oligosaccharides with a degree of polymerization of 4–6 were hydrolyzed by the enzyme, and the same endo-products, cellotriose, cellobiose and glucose, were produced from these oligosaccharides. Neither cellotriose nor cellobiose was hydrolyzed by the enzyme. The enzyme digested filter paper and sea lettuce to produce cellobiose, cellotriose and glucose as major products. Although amino acid sequence homology of the enzyme with termite cellulases and the presence of oligosaccharides in the enzyme suggested that the enzyme is produced by krill itself, further analysis is necessary.

A series of oligomeric glycans can be extracted from the cell walls of developing cotton fibers with weak acid. Glycans that produce similar profiles on high pH anion chromatography with pulsed amperometric detection (HPAEC-PAD) are also found in a protein complex extracted from developing fibers and in amorphous aggregates found in association with immature fibers in developing, but not in mature cotton bolls. The quantity and composition of the glycans recovered from the carbohydrate-protein complex varies significantly with the time of day when the bolls are harvested. This diurnal variation is consistent with the hypothesis that secondary cell walls are deposited primarily at night. Incubation of re-hydrated cotton fibers in the presence of exogenous oligosaccharides, myo-inositol and glycerol substantially alters the apparent quantity of the oligomers extracted from the fibers. The same and similar glycans have also been extracted from cotton fabric, marine algae, various paper products and wood. While many of the oligomers isolated from the various cellulose sources display the same peaks by HPAEC-PAD, the specific number of oligomers and their relative quantities appear unique for each source of cellulosic material. Oligomeric glycans, as described in the preceding, are present in all cellulose sources that have been investigated. Their relative abundance changes in response to source, stage of development and other physiological variables. We hypothesize that the glycans are intermediates in the biological assembly of cellulose, and that their incorporation in cellulose is mediated by physicochemical and enzymatic mechanisms.

Carbohydrate analysis is an important source of the information required for understanding and control of pulp and paper processes. The behavior of cellulose and hemicelluloses in the process, carbohydrate–lignin interactions, and the enzymatic treatment of fibers are examples of situations for which reliable, fast, qualitative, and quantitative methods are required. New uses of lignocellulosic material have further increased the need for carbohydrate analysis. This review collates and summarizes the most important findings and approaches in the analysis of wood-based carbohydrates by use of capillary zone electrophoresis and provides an analysis of the effect of different conditions on the separation, showing the advantages and limitations of the methods used. It provides guidelines for achieving higher quality and improved separation efficiency in carbohydrate analysis.

Asparagine-linked sugar chains were quantitatively released from chimpanzee, Rhesus monkey and rat IgA proteins as oligosaccharides by hydrazinolysis, converted to radioactive oligosaccharides by reduction with NaB3H4, and separated into neutral and two acidic fractions by paper electrophoresis. The acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. However, the content of N-acetyl and N-glycolyl neuraminic acids was different among three species. The neutral and sialidase-treated acidic oligosaccharides were fractionated by Bio-Gel P-4 column chromatography in combination with linkage-specific sequential exoglycosidase digestion. Although IgA molecules from these species have mainly biantennary complex-type sugar chains, the contents of fucose and bisecting N-acetylglucosamine residues displayed marked species differences. In addition to these sugar chains, a small amount of the high mannose-type sugar chains was detected in chimpanzee and rat, but not in Rhesus monkey IgA. These results indicated that the processing of asparagine-linked sugar chains of IgA is different in each species.

The cellulases of Streptomyces thermodiastaticus (strain 2Sts) and thermomonospora fusca (strain 190Th) were produced with carboxymethyl-cellulose (CMC) serving as the carbon source during growth. Both cellulases act by random internal hydrolysis of the CMC chain, producing cellobiose, glucose, and intermediate length oligosaccharides. Cellobiase was not detected in culture filtrates produced under these conditions.