Investigation of the interaction between enzyme and inhibitor by the combination of chemical modification, electrospray ionization mass spectrometry and frit-fast atom bombardment liquid chromatography/mass spectrometry

Abstract

The interaction between enzyme and its inhibitor, hen egg-white lysozyme and tri-N-acetylglucosamine (NAG3), was studied by the combination of chemical modification, enzymatic digestion, electrospray ionization mass spectrometry and frit-fast atom bombardment liquid chromatography/mass spectrometry. Chemical modification of amino groups, carboxyl groups, and indole groups was carried out independently. In the absence of NAG3, the carboxyl group in Asp 101 was modified by glycinamidation, and the indole group in Trp 62 was modified by Koshland reagent. In the presence of NAG3, the degree of modification of Asp 101 and Trp 62 decreased. It is suggested that Asp 101 and Trp 62 are involved in the interaction with NAG3. The result is consistent with the one obtained by x-ray crystallography. It is indicated that the combination of chemical modification and mass spectrometry may be effective for the investigation of the binding reaction of enzyme to inhibitor and of protein-protein interaction.

The role of the 6 lysines and the terminal amine of escherichia coli single‐strand binding protein in its binding of single‐stranded DNA

“…Recent advances in mass spectrometry have made it possible to assess the reactivities of amino acid side chains without the use of radioactive materials (Qin et al, 1992;Akashi et al, 1993;Hasan et al, 1993;Glocker et al, 1994). Due to the high specificity of mass spectrometry, modified peptides can be detected and identified even when isolation is incomplete.…”

The use of mass spectrometry to examine the formation and hydrolysis of the phosphorylated form of phosphoglycerate mutase

“…The advent of electrospray mass spectrometry with its ability to measure molecular masses with a precision of +0.01% has made it much easier to detect and characterise both post-translational and chemical modifications of proteins [1][2][3]. The introduction of the phospho group (-OPO 2-) in place of-H would lead to a mass increase of 78 units and thus be readily detectable.…”

Towards integrative structural mass spectrometry: Benefits from hybrid approaches

“…Although NMR has higher resolution, MS had the clear advantage in terms of sensitivity, sample and time consumption, and the size limit of the proteins studied. At the same period, covalent labeling coupled to LC-MS started to emerge as a promising technique to probe protein surface topology [16][17][18][19][20]. A variety of chemical modifications (diethylpyrocarbonate, acetylation, carboxylation, oxidation) (reviewed in [21]) can be used to probe the surface accessibility of different amino acids.…”