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|Title:||Mass Spectrometric Studies of Enzymatic Reactions|
|Author(s):||Murawski, Sandra Lou|
|Department / Program:||Chemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The use of mass spectrometry for the study of proteolytic enzymatic reactions was evaluated. Two types of soft ionization were employed, electrohydrodynamic ionization and fast atom bombardment.
Initial work with electrohydrodynamic ionization mass spectrometry involved modifying the ion source to permit analysis of aqueous and partially aqueous solutions. This was accomplished mainly through the use of emitters with small capillary openings.
An electrohydrodynamic mass spectral survey of twenty common amino acids revealed that all could be sampled as protonated or sodiated adducts, with little or no fragmentation. Electrohydrodynamic sampling efficiencies for amino acids in a mixture were not identical. Quantitation would therefore require use of an internal standard.
Hydrolysis product ions were detected in the electrohydrodynamic mass spectra of a reaction mixture containing carbobenzoxy-L-phenylalanyl-L-leucine and carboxypeptidase Y. Alternate solvents were evaluated in efforts to improve analyte sampling efficiency. More work is needed to fully evaluate the potential of electrohydrodynamic mass spectrometry for real-time enzymatic reaction monitoring.
Fast atom bombardment mass spectrometry with moving belt sample introduction was used to follow peptide hydrolyses in situ. Kinetic parameters were estimated for the tryptic hydrolyses of (alpha)-N-p-tosyl arginine methyl ester and serum thymic factor. Several sequential reactions during the tryptic hydrolysis of mastoparan and the hydrolysis of bradykinin by carboxypeptidase Y were monitored by the real-time fast atom bombardment mass spectrometry method. Use of this method to monitor enzymatic sequencing of small peptides (MW < 2000) should be possible.
Fast atom bombardment mass spectrometry was used to examine several cyanogen bromide peptides of putidaredoxin. Full scan mass spectra, linked scans on the parent ion, and peptide mapping using trypsin were useful in identifying the peptides. These methods should be valuable in characterizing similar peptides with unknown structures.
Acetylcholine and choline levels in human cerebrospinal fluid were estimated using fast atom bombardment mass spectrometry and stable isotope dilution. Levels ranged from 0 to 440 pmole/mL for acetylcholine and from 2.1 to 3.6 nmole/mL for choline. These values agree well with levels determined by other investigators using a variety of methods.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1987.
|Date Available in IDEALS:||2014-12-15|