The rate of oxygen exchange of each epimer as a function of epimerization of 15(i?)-methylprostaglandin E2 (R) and 15(S)-methylprostaglandin E2 (S) (tertiary alcohols) in 180-enriched water has been determined by LC and GC-MS methods. The relative rates indicate that exchange of the C-15 hydroxyl group is more rapid than the change in configuration at C-15. These data are consistent with a mechanism for epimerization in which the initially formed carbonium ion formally retains the configuration of starting material. A significant percentage of the initially formed epimerized product does not exchange with the ,80-enriched water. This result indicates that, in some cases, the departing water molecule ultimately bonds the carbonium ion from the opposite face. A reaction scheme is proposed in which the waters of solvation exchange with the departing water more rapidly than the initially formed carbonium ion epimerizes.
Ion-spray ionization mass spectrometry with gentle conditions for solvent removal has been reported as a useful tool for detection of high-affinity noncovalent complexes of biological relevance formed in solution. Two main objectives of this study were (i) to find whether other types of electrospray ionization (ESI) sources, e.g. where the solvent is removed with the help of heat (thermally assisted electrospray), could be utilized for detection of noncovalent biological complexes of high and low affinity and (ii) to find whether ESI-MS can be used for detection of the association of bovine serum albumin (BSA) with biologically active peptides. Using a well-defined high-affinity association of FK506 with its binding protein (FKBP) as model system we proved that ESI-MS with thermally assisted interphase can be used for detection of the FK506-FKBP complexes in a similar way as was previously shown for electrospray mass spectrometry (Ganem et al., J. Am. Chem. Soc. 113, 6294 (1991)). In mixtures of BSA with a 9-10 molar excess of biologically active peptides, such as growth hormone releasing factor (GRF), glucagon, bradykinin or insulin in ammonium acetate at pH 7.5, complexes with a ratio of 1:1, 1:2 and in some cases 1:3 were detected. On the other hand, these complexes disappeared upon acidification, pointing to their noncovalent nature.(ABSTRACT TRUNCATED AT 250 WORDS)
A Beckman P/ACE 2050 high-performance capillary electrophoresis (HPCE) instrument has been interfaced with a Vestec electrospray ionization (ESI) mass spectrometer for the analysis of recombinant proteins. Peak resolution is not compromised by coupling HPCE to an ESI mass spectrometer. Recombinant bovine and porcine somatotropins (rbSt and rpSt) were used as model proteins. The standard curve of the capillary zone electrophoresis (CZE) method with UV detection for the determination of rpSt is linear in the range of 7-300 fmol with theoretical plates of approximately 410,000 m-1. The relative standard deviation for the rpSt peak migration time is less than 1%. The multiply-charged ion clusters obtained in the CZE-ESI mass spectrum for a sample of rpSt ranged from mlz 1363.2 (the cluster with 16 charges) to 1982.5 (the cluster with 11 charges). The average molecular weights of 21,812.6 and 21,798.3 for a sample of rbSt and rpSt determined in this study were nearly identical to the theoretical values of 21,812.0 and 21,797.9, respectively. Detection limit of the CZE-ESI mass spectrometer is approximately 100 fmol. The CZE method separated mono- and dideamidated species and monoacetylated compounds while the ESI mass spectrometer detected an analogue and a truncated homologue of rpSt comigrating with the major peak. The presence of mono- and dioxidized homologues was also detected in the major peak of some rbSt and rpSt samples. These data clearly indicated that, individually, both CZE and ESI mass spectrometric methods could not detect all impurities. Coupling of the HPCE Instrument and the ESI mass spectrometer enhances analytical capabilities of both tools for rapid characterization of recombinant proteins.
Thermospray liquid chromatography/mass spectrometry using a double-focusing magnetic sector mass spectrometer has been studied. The quality of the thermospray mass spectra were often a function of the solvent mixture used, and use of gradients affected the quality of some thermospray mass spectra. High resolution mass measurements using peak matching gave excellent results for pure compounds and mixtures which did not have 13C isotope interferences. When 3C isotope interference was a problem high-performance liquid chromatographic separation together with magnetic scanning was used.
A high performance liquid chromatographic assay of trace amounts of 5-/rans-PGE2 in PGE2 has been developed. This assay, capable of assaying as little as 0.20% 5-frans-PGE2 with a precision of ±10%, is based on the chromatographic separation of the p-nitrophenacyl esters of these compounds on a silver ion-loaded cation exchange resin. This chromatographic column resolves the PGE2-p-nitrophenacyl esters from those of PGA2, PGB2j PGF1a, and PGF2a.
The electrospray mass spectra of several standard proteins were recorded and their molecular weights determined. These were compared to Literature values obtained by other laboratories. The agreement was quite good. Seven recombinant growth hormones were then investigated by this technique. The determined molecular weights were in agreement with the theoretical values. Differentiation between six of the seven analogs could be made on the basis of the molecular weight determinations. Two of these analogs differed in molecular weight by only 1 Da and the accuracy of the mass measurements was not sufficient for distinguishing these two homologous proteins. The influence of solvent on the mass accuracy determinations was studied. It appears that solvents form clusters with the proteins in the electrospray ionization method. These clusters broaden the peaks corresponding to the multiply charged ions of the proteins and make the determination of centroids more difficult.
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