STRATEGY FOR THE CHARACTERIZATION OF THE GLYCOPROTEIN ENDOGLUCANASE-I ISOLATED FROM TRICHODERMA-REESEI - COMBINATION OF PLASMA DESORPTION AND UV LASER DESORPTION TIME-OF-FLIGHT MASS-SPECTROMETRY

The first results are reported of a fast time-of-flight mass spectrometry (TOF-MS)-based strategy for characterizing the large glycoprotein endoglucanase I using chemical/ enzymatic cleavage reactions and two soft ionization techniques, Cf252 plasma desorption (PD) and UV laser desorption (UVLD). The glycoprotein was isolated from the extracellular culture fluid of the fungus Trichoderma reesei grown on cellulose as a carbon source. The glycoprotein was purified by various chromatographic steps. In this strategy, as first step the molecular weight of the intact glycoprotein was determined by positive ion matrix-assisted UVLD-MS. The value obtained for endoglucanase I was 52 110 daltons. From the DNA-derived amino acid sequence, the molecular weight of the protein moiety was calculated. The mass difference between the UVLD-MS-determined molecular weight and the protein moiety gave the carbohydrate content. The next step was the confirmation of the amino acid sequence deducted from the nucleotide sequence of the gene. The glycoprotein was cleaved chemically with cyanogen bromide (CNBr) and the unseparated peptide and glycopeptide mixture was adsorbed on a nitrocellulose-covered target for PD-MS. The positive ion spectra (washed and unwashed) revealed a number of protonated molecular ions, which partly matched the calculated [M + H]+ ions of CNBr peptides predicted by the gene sequence; 39% of the amino acid sequence was covered by the peptides. Then carboxypeptidase Y was added to the nitrocellulose-adsorbed peptides. The C-terminal removal of amino acids was monitored by PD-MS. The PD mass spectrum showed several protonated molecular ions and the measured mass differences obtained were used to obtain partial amino acid sequence information. These results gave further support of the correctness of the DNA-derived amino acid sequence. This new strategy permitted the rapid characterization of endoglucanase I with respect to molecular weight, carbohydrate content and partial amino acid verification.