Characterization of a mixture of lobster digestive cysteine proteinases by ionspray mass spectrometry and tryptic mapping with LC—MS and LC—MS—MS

• Published in: International journal of mass spectrometry and ion processes 1991-12, Vol.111, p.317-353
• Main Authors: Thibault, P., Pleasance, S., Laycock, M.V., Mackay, R.M., Boyd, R.K.
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases
• ISSN: 0168-1176; 1873-2801
• DOI: 10.1016/0168-1176(91)85063-R

An inseparable mixture of two cysteine proteinases, isolated from the digestive tract of the American lobster, was investigated by ionspray mass spectrometry (ISP-MS), using a combination of infusion of intact proteins with on-line liquid chromatography—mass spectrometry (LC—MS) and LC—MS—MS analyses of tryptic digests. These data were interpreted by comparisons with predictions from results of molecular cloning of cysteine-proteinase-encoding messenger RNA sequences previously isolated from the lobster hepatopancreas. Investigations of the numbers of free thiol groups and of disulfide bonds were made by measuring the molecular weights of the alkylated proteins with and without prior reduction of disulfide bonds, and comparison with the corresponding data for the native proteins. Identification of tyrptic fragment peptides containing cysteine residues was facilitated by comparing LC—MS analyses of tryptic digests of denatured and of denatured and alkylated proteins, since such tryptic peptides are subject to shifts in both mass and retention time upon reduction and alkylation. Confirmation of amino acid sequences was obtained from fragment ion spectra of each tryptic peptide (alkylated or not) as it eluted from the column. Acquisition of such on-line LC—MS data was possible through use of the entire effluent from a standard 1 mm high performance liquid chromatography (HPLC) column by an IonsSpray ® LC—MS interface (pneumatically assisted electrospray).