Edwin J. Bakx

A xylogalacturonan subunit present in the modified hairy regions of apple pectin.

Abstract The high molecular weight fraction (fraction A) of the modified hairy regions (MHR) from apple cell walls was treated by rhamnogalacturonase (RGase) after saponification and after deacetylation by rhamnogalacturonan acetylesterase (RGAEase). Three fractions could be recognized by size-exclusion chromatography: rhamnogalacturonan oligomers, residual stubs of the rhamnogalacturonan backbone rich in arabinan side-chains, and a fraction rich in xylose and galacturonic acid. The “xylogalacturonans” obtained after saponification and after enzymic deacetylation had rather similar sugar compositions (xylose:galacturonic acid ratios of 0.4–0.9) and molecular weights. After saponification, the xylogalacturonan was eluted as a single peak on anion-exchange chromatography whereas three peaks were obtained when the MHR was deacetylated by RGAEase, indicating variations in the degrees of methylation. One of the methyl-esterified xylogalacturonan fractions was characterized by NMR spectroscopy: the xylose residues were β-(1 → 3)-linked to some of the galacturonic acid residues within a rather high molecular weight xylo-(1 → 4)-α-galacturonan and a degree of methylation of 39 was calculated. The methyl esters were found to be equally divided between the substituted and unsubstituted galacturonosyl residues.

Peptides are Building Blocks of Heat-Induced Fibrillar Protein Aggregates of β-Lactoglobulin Formed at pH 2

The proteinaceous material present in beta-lactoglobulin fibrils formed after heating (20 h at 85 degrees C) at pH 2 was identified during this study. Fibrils were separated from the nonaggregated material, and the fibrils were dissociated using 8 M guanidine chloride and 0.1 M 1,4-dithiothreitol (pH 8). Characterization of the different fractions was performed using thioflavin T fluorescence, high-performance size-exclusion chromatography, reversed-phase HPLC, and mass spectrometry (MALDI-TOF). Beta-lactoglobulin was found to be hydrolyzed into peptides with molecular masses between 2000 and 8000 Da, and the fibrils were composed of a part of these peptides and not intact beta-lactoglobulin. The majority of the peptides (both aggregated and nonaggregated) were a result from cleavage of the peptide bonds before or after aspartic acid residues. Explanations for the presence of certain peptide fragments in the fibrils are the hydrophobicity, low charge, charge distribution, and capacity to form beta-sheets.

Okra pectin contains an unusual substitution of its rhamnosyl residues with acetyl and alpha-linked galactosyl groups

The okra plant, Abelmoschus esculentus (L.) Moench, a native plant from Africa, is now cultivated in many other areas such as Asia, Africa, Middle East, and the southern states of the USA. Okra pods are used as vegetables and as traditional medicines. Sequential extraction showed that the Hot Buffer Soluble Solids (HBSS) extract of okra consists of highly branched rhamnogalacturonan (RG) I containing high levels of acetyl groups and short galactose side chains. In contrast, the CHelating agent Soluble Solids (CHSS) extract contained pectin with less RG I regions and slightly longer galactose side chains. Both pectic populations were incubated with homogeneous and well characterized rhamnogalacturonan hydrolase (RGH), endo-polygalacturonase (PG), and endo-galactanase (endo-Gal), monitoring both high and low molecular weight fragments. RGH is able to degrade saponified HBSS and, to some extent, also non-saponified HBSS, while PG and endo-Gal are hardly able to degrade either HBSS or saponified HBSS. In contrast, PG is successful in degrading CHSS, while RGH and endo-Gal are hardly able to degrade the CHSS structure. These results point to a much higher homogalacturonan (HG) ratio for CHSS when compared to HBSS. In addition, the CHSS contained slightly longer galactan side chains within its RG I region than HBSS. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated the presence of acetylated RG oligomers in the HBSS and CHSS enzyme digests and electron spray ionization-ion trap-mass spectrum showed that not only galacturonosyl residues but also rhamnosyl residues in RG I oligomers were O-acetylated. NMR spectroscopy showed that all rhamnose residues in a 20kDa HBSS population were O-acetylated at position O-3. Surprisingly, the NMR data also showed that terminal alpha-linked galactosyl groups were present as neutral side chain substituents. Taken together, these results demonstrate that okra contained RG I structures which have not been reported before for pectic RG I.

A rapid screening method for prenylated flavonoids with ultra-high-performance liquid chromatography/electrospray ionisation mass spectrometry in licorice root extracts.

Due to their substitution with an isoprenoid group, prenylated flavonoids have an increased affinity for biological membranes and target proteins, enhancing their potential bioactivity. Although many prenylated flavonoids have been described, there are no methods that specifically screen for their presence in complex mixtures, prior to purification. We describe a method based on ultra-high-performance liquid chromatography (UHPLC) with electrospray ionisation mass spectrometry (ESI-MS) that allows rapid screening for prenylated flavonoids in multi-component plant extracts. Identification of the prenylated flavonoids is based on screening for neutral losses of 42 u and 56 u in the positive-ion mode MS(2) and MS(3) spectra within the MS chromatograms. In addition, this method discriminates between a prenyl chain and a ring-closed prenyl (pyran ring), based on the ratio of the relative abundances of the ions that lose 42 u and 56 u (42:56). The application of this screening method on a 70% aq. ethanol, ethanol and ethyl acetate extract of the roots of Glycyrrhiza glabra indicated the presence of 70 mono- and di-prenylated flavonoids. In addition, of each prenylated flavonoid the type of prenylation, chain or pyran ring was determined.

Structural Characterisation by ESI-MS of Feruloylated Arabino-oligosaccharides Synthesised by Chemoenzymatic Esterification

The chemoenzymatic synthesis of feruloylated arabino-oligosaccharides has been achieved, using a feruloyl esterase type C from Sporotrichum thermophile (StFaeC). The structure of the feruloylated products was confirmed by ESI-MS(n).

Identification of novel isomeric pectic oligosaccharides using hydrophilic interaction chromatography coupled to traveling-wave ion mobility mass spectrometry

Separation and characterization of complex mixtures of pectic oligosaccharides still remains challenging and often requires the use of multiple analytical techniques, especially when isomeric structures are present. In this work, it is demonstrated that the coupling of hydrophilic interaction chromatography (HILIC) to traveling-wave ion mobility mass spectrometry (TWIMMS) enabled the simultaneous separation and characterization of complex mixtures of various isomeric pectic oligosaccharides. Labeling of oligosaccharides with 3-aminoquinoline (3-AQ) improved MS-ionization efficiency of the oligosaccharides and reduced the complexity of the product ion mass spectra, without losing resolution of the HILIC separation. In addition, labeling enabled quantification of oligosaccharides on molar basis using in-line fluorescence detection. Isomeric structures were distinguished using TWIMMS. The 3-AQ-HILIC-TWIMMS method was used to characterize a series of isomeric sugar beet rhamnogalacturonan I derived oligosaccharides carrying a glucuronic acid substituent. Thereby, some novel structural features were identified for the first time: glucuronic acid was attached to O-3 or to O-2 of galacturonic acid residues and a single galacturonic acid residue within an oligomer could contain both an acetyl group and a glucuronic acid substituent.

The structure of an alternative wall teichoic acid produced by a Lactobacillus plantarum WCFS1 mutant contains a 1,5-linked poly(ribitol phosphate) backbone with 2-α-d-glucosyl substitutions

A tagF1-tagF2 deletion mutant of Lactobacillus plantarum lacks poly(glycerol phosphate) polymerase activity required for glycerol-type wall teichoic acid (WTA) biosynthesis. The mutant activates an alternative genetic locus, tarIJKL, encoding the enzymes for nucleotide activation and incorporation of ribitol in the WTA backbone polymer. This alternative ribitol-type WTA backbone and its repeating unit were isolated and characterized by HPAEC, UPLC-MS, NMR spectroscopy, and MALDI-TOF MS, using synthetic molecules as references. The structure was established as 1,5-linked poly(ribitol phosphate) which was substituted at the C-2 hydroxyl group of the ribitol residue with α-D-glucosyl at a frequency of 28%.

DIFFERENCES IN THE METHYL ESTER DISTRIBUTION OF PECTINS

Both commercial pectin preparations as well as pectins as present in plant cell walls are very complex and heterogeneous molecules. Although the relative amount of the rhamnogalacturonan structural element may very significant for pectins from different origine, most pectins have in common that homogalacturonans forms the major part of the molecule. These homogalacturonan segments partly determine the functional properties of the pectin like its thickening and gelling properties. Especially the level of methyl esters and their distribution over the galacturonan backbone is of major importance. Many effort has been directed to the reveal the pattern of methyl ester distribution.

Characterisation of 3-aminoquinoline-derivatised isomeric oligogalacturonic acid by travelling-wave ion mobility mass spectrometry

RATIONALE Mass spectrometry has become a useful technique for elucidating the chemical structures of oligosaccharides. The combined use of chromatography and mass spectrometry for the separation and identification of oligosaccharides has shown much progress in recent years. However, no powerful method has yet been developed to quickly identify isomeric oligosaccharides in complex mixtures. METHODS A rapid travelling-wave ion mobility mass spectrometry (TWIMS-MS) method was developed for the identification of various isomeric oligogalacturonic acids in mixtures and determined their structures, using 3-aminoquinoline (3-AQ) as a labelling agent. RESULTS TWIMS successfully distinguished isomeric oligogalacturonic acids of various degrees of polymerisation (DPs) and levels of methyl-esterification. After derivatisation by 3-AQ, isomeric oligosaccharides of galacturonic acid, with the DP ranging from 2 to 9 and the number of methyl esters ranging from 1 to 5, were identified by 3-AQ-TWIMS-MS. The isomeric oligosaccharides with varying sites of methyl ester substitution were identified by the post-fragmentation mode of TWIMS using 3-AQ labelling to obtain simplified mass spectra. CONCLUSIONS Using the 3-AQ-TWIMS-MS method, the precise distribution of methyl esters within the pectin molecule and isomeric oligogalacturonic acids after enzyme degradation was determined. Simplified product ion mass spectra and precise analysis of the isomers were achieved by labelling 3-AQ at the reducing end of the oligosaccharides. Series of methyl-esterified galacturonic acid oligomers have predictable drift times, depending on the precise position of the methyl ester.