No abstract

It was previously revealed [Yamaguchi, H. and Uchida, M. (1996) J. Biochem. 120, 474-477] that both intra- and extramolecular high-mannose type Asn-glycans promote the renaturation of reductively denatured bovine pancreatic RNases A and B under oxidation conditions. To characterize the conformational changes of the polypeptides during the renaturation promoted by the intramolecular Asn-glycans, RNase B was compared with its nonglycosylated form, RNase A, as to the features of the regeneration from their reductively denatured species under Cu2+-catalyzed oxidation conditions. The refolding intermediates of RNase B, as compared with those of RNase A, seemed to contain much less impaired disulfide linkages. In agreement with this finding, the proper refolding of RNase B was much faster than that of RNase A, as revealed by the intrinsic fluorescence and 1-anilino-8-naphthalenesulfonate binding of the refolding intermediates. Such a promoting effect was also observed for extramolecular Asn-glycans of the complex as well as of the high-mannose type. In contrast, common mono-, oligo-, and polysaccharides, but not yeast mannan, exhibited much lower stimulatory effects on the oxidative refolding of RNase A.

Ion trap/time-of-flight hybrid mass spectrometers are powerful tools for the detailed structural analysis of modified peptides. We have analyzed Met-Lys-bradykinin modified with deoxycholate at the amino-terminus or the epsilon-amino group as model peptides. These two modified peptides produced fragment ions with the same nominal but different exact masses in tandem mass spectrometry with low-energy collision-induced dissociation. Accurate high-resolution analysis coupled with MS(3) allowed us to distinguish the deoxycholate modification sites in the modified peptides.

Hybrid quadrupole ion trap/time-of-flight mass spectrometry (QIT/ TOFMS) was applied for detection of ultra fast liquid chromatography (UFLC). The system performance for fast qualitative analysis was evaluated using test drugs. The test drugs were separated on a C 18 column (30 mm  2.0 mm I.D., particle size: 2.2 mm) with fast linear gradient elution using 0.1% formic acid and acetonitrile as mobile phase. The flow rate was set at 0.5 mL/min and the analysis cycle time was 4.5 min. Relative standard deviations (RSDs) of retention time and peak area for each drug (200 pg each) were 0.2% or better, lower than 3%, respectively. Mass accuracy of each compound was found to be 3.2 ppm or better. Rapid positive to negative polarity switching mode was demonstrated, showing good mass accuracy below 5 ppm. Moreover, an MS 3 measurement was carried out and the formulae of compounds were confirmed using formula prediction software.

Metabolite identification is a critical step in drug discovery and development. Finding metabolites by comparing sample data with control data is both di$cult and tedious, despite the various software applications available. The objective of this work was to detect metabolites from liquid chromatography coupled mass spectrometry/mass spectrometry (MS/MS) data by using only the product ion mass spectrum. Molecules that have similar structures can be selected easily and rapidly from auto MS/MS data using partial least squares (PLS) for the product ion mass spectra. Major metabolites of verapamil from the fraction in the presence of Phase I co-factors are well known. Automated ion trap MS/MS data-dependent acquisition with PLS was successfully applied and the major known metabolites and one unexpected metabolite were detected. Their structures are elucidated in this paper. It is suggested that automated ion trap MS/MS data-dependent acquisition with PLS can be a useful tool in detecting metabolites and elucidating their structure.