Chitin/chitosan oligosaccharides composed of 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) and/or 2-amino-2-deoxy-D-glucopyranose (GlcN) were prepared by chemical degradation of chitin or chitosan and separated by gel permeation chromatography. Oligosaccharides obtained after enzymatic hydrolysis of chitosan [F(A) 0.19] with a fungal chitinase were derivatized by reductive amination with 2-aminoacridone and sequenced by matrix-assisted laser desorption ionization time-of-flight postsource decay (PSD) mass spectrometry (MS). The sequence of a trimer, D1A2, was established as D-A-A. The composition of a hexamer D3A3 was ca. 65% D-A-D-D-A-A and 35% D-D-A-D-A-A. The PSD MS of a nonamer D5A4-amac revealed four isobaric species D-X-Y-D-X-Y-D-A-A, where A is GlcNAc, D is GlcN, and X and Y (X not equal Y) are mutually either D or A. This structure motif was also observed in a dodecamer D7A5 which was composed of eight isobaric sequences of the general formula (D-X-Y)(3)-D-A-A.

Heterochitooligosaccharides possess interesting biological properties. Isobaric mixtures of such linear heterochitooligosaccharides can be obtained by chemical or enzymatic degradation of chitosan. However, the separation of such mixtures is a challenging analytical problem which is so far unresolved. It is shown that these isobaric mixtures can be sequenced and quantified simultaneously using standard derivatization and multistage tandem mass spectrometric techniques. A linear ion trap mass spectrometer equipped with a vacuum matrix-assisted laser desorption ionization (vMALDI) source is used to perform MS2 as well as MS3 experiments.

No abstract

Oligosaccharides are of current interest as targets for the development of novel pharmaceuticals and plant-growth regulators.[1] For example, so-called heterochitooligosaccharides, which are composed of N-acetylglucosamine (GlcNAc or A) and glucosamine (GlcN or D), exhibit various biological activities, such as promotion of chondrocyte growth in cell culture, morphogenetic activity in vertebrates, and elicitor action in plants.[2] The entities used for biological studies are usually prepared by enzymatic hydrolysis of the aminoglucan chitosan, clearly yielding intractable, complex mixtures of aminoglucan oligomers. These oligomers differ in their degree of polymerization (DP; number of monomer units), as well as in the mole fraction of A residues (that is, homologues) and in the sequences of D and A residues (that is, isomers).Despite the fact that several protein-ligand complexes containing GlcNAc homooligomers or enzyme inhibitors have been investigated by protein crystallography [2, 3] and 1 H NMR spectroscopy, [4] the molecular mechanisms of the biological actions of heterochitooligosaccharides remain essentially unknown as a result of a lack of structural information and the fact that pure ligands are not available. One option for obtaining more information is the use of mass spectrometry (MS), which may be employed for the detection and chemical/structural analysis of noncovalent proteincarbohydrate complexes. In principle, MS-based techniques could provide information on binding stoichiometry and identification of the ligand by fragmentation. [5,6] We observed earlier that mixtures of A n D m heterochitooligosaccharides with a mole fraction of A units (F A ) of 0.5 and a DP of 5 or 6 contain inhibitory compounds with high affinity for the chitinolytic enzyme chitinase B (ChiB), from the soil bacterium Serratia marcescens.[5] The apparent IC 50 value (inhibitory concentration 50 %) was about 15 mm, with (GlcNAc) 2 -4-methylumbelliferone (A 2 -MU) as the substrate (Michaelis constant of A 2 -MU, K m = 30 mm [7] ). Assuming that not all components are inhibitors, the IC 50 value of the active component must indeed be lower, possibly in the sub-mmol range. The challenge was now to identify a high-affinity binding ligand from a complex oligosaccharide mixture.Herein, we report on the specificity and stoichiometry of enzyme-ligand recognition, by using (+) nanoelectrospray ionization (nanoESI) MS for analysis of the formation of a noncovalent protein-ligand complex. A novel method based on nanoESI quadrupole time-of-flight collision-induced dissociation tandem mass spectrometry (nanoESI-QTOF-CID MS/MS) was employed for top-down sequencing of the complex-generating fragment ions of the bound ligand. The MS-based method provides an excellent tool for the study of biological complexes, [6,8,9] especially when protein crystallography or NMR spectroscopy cannot be applied.The choice of nanoESI-QTOF MS was guided by the advantage of using low flow rates for sample infusion [10] in a hybrid system, which combines the...

Oligosaccharides are of current interest as targets for the development of novel pharmaceuticals and plant-growth regulators.[1] For example, so-called heterochitooligosaccharides, which are composed of N-acetylglucosamine (GlcNAc or A) and glucosamine (GlcN or D), exhibit various biological activities, such as promotion of chondrocyte growth in cell culture, morphogenetic activity in vertebrates, and elicitor action in plants.[2] The entities used for biological studies are usually prepared by enzymatic hydrolysis of the aminoglucan chitosan, clearly yielding intractable, complex mixtures of aminoglucan oligomers. These oligomers differ in their degree of polymerization (DP; number of monomer units), as well as in the mole fraction of A residues (that is, homologues) and in the sequences of D and A residues (that is, isomers).Despite the fact that several protein-ligand complexes containing GlcNAc homooligomers or enzyme inhibitors have been investigated by protein crystallography [2, 3] and 1 H NMR spectroscopy, [4] the molecular mechanisms of the biological actions of heterochitooligosaccharides remain essentially unknown as a result of a lack of structural information and the fact that pure ligands are not available. One option for obtaining more information is the use of mass spectrometry (MS), which may be employed for the detection and chemical/structural analysis of noncovalent proteincarbohydrate complexes. In principle, MS-based techniques could provide information on binding stoichiometry and identification of the ligand by fragmentation. [5,6] We observed earlier that mixtures of A n D m heterochitooligosaccharides with a mole fraction of A units (F A ) of 0.5 and a DP of 5 or 6 contain inhibitory compounds with high affinity for the chitinolytic enzyme chitinase B (ChiB), from the soil bacterium Serratia marcescens.[5] The apparent IC 50 value (inhibitory concentration 50 %) was about 15 mm, with (GlcNAc) 2 -4-methylumbelliferone (A 2 -MU) as the substrate (Michaelis constant of A 2 -MU, K m = 30 mm [7] ). Assuming that not all components are inhibitors, the IC 50 value of the active component must indeed be lower, possibly in the sub-mmol range. The challenge was now to identify a high-affinity binding ligand from a complex oligosaccharide mixture.Herein, we report on the specificity and stoichiometry of enzyme-ligand recognition, by using (+) nanoelectrospray ionization (nanoESI) MS for analysis of the formation of a noncovalent protein-ligand complex. A novel method based on nanoESI quadrupole time-of-flight collision-induced dissociation tandem mass spectrometry (nanoESI-QTOF-CID MS/MS) was employed for top-down sequencing of the complex-generating fragment ions of the bound ligand. The MS-based method provides an excellent tool for the study of biological complexes, [6,8,9] especially when protein crystallography or NMR spectroscopy cannot be applied.The choice of nanoESI-QTOF MS was guided by the advantage of using low flow rates for sample infusion [10] in a hybrid system, which combines the...