Identification of a High‐Affinity‐Binding Oligosaccharide by (+) Nanoelectrospray Quadrupole Time‐of‐Flight Tandem Mass Spectrometry of a Noncovalent Enzyme–Ligand Complex

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...

Substrate positioning in chitinase A, a processive chito-biohydrolase fromSerratia marcescens

“…Screening for complex formation and topdown analysis of detected complexes were performed as described earlier [25]. For all experiments the following parameters were used: the ion-source temperature was set at 80°C, desolvation gas was used at a flow rate of 100 l/h, a potential of 900 V was applied to the capillary tip, and the sampling cone voltage was set to 80 V. For collision-induced dissociation (CID)-analysis, the parent ion was selected by the first quadrupole where about ten scans were acquired under the following conditions: the low-and high-mass resolution of the quadrupole for isolation where set to 0; the ion acceleration voltage was set to 4 eV.…”

“…Previously, it has been suggested that non-productive binding of partially de-acetylated CHOS is due to binding of a deacetylated sugar to the -1 subsite [25]. Such binding would be non-productive because of the involvement of the acetamidogroup of the -1 sugar in catalysis, [30].…”

Reliable Determinations of Protein–Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?

“…These features enable the determination of both the macroscopic and microscopic K a values for sequential binding of L to P. As a result, ESI-MS is ideally suited for characterizing allosteric binding. The ESI-MS assay also naturally lends itself to monitoring and quantifying protein-ligand interactions in solutions containing mixtures of ligands and/or proteins [25][26][27][28][29][30][31][32][33]. Not surprisingly, an emerging ESI-MS application is screening libraries of compounds against target proteins to identify specific interactions.…”

Perspectives of Chitin Deacetylase Research

“…Besides that, for a complete analysis, a large amount of expensive enzymes and chitin oligomers are required. Compared with the above method, MS n has been shown to be a powerful technique for structural elucidation of hetero-COS (Bahrke et al 2002, Cederkvist et al 2006, Haebel et al 2007, Issaree 2008, which was recently reviewed by Peter and Eberlin (2010). Bahrke summarized different techniques employed for the quantitative analysis of mixtures containing heterochitooligomers, homologs, and isomers (Fig.…”