Novel methodologies for the characterization and identification of proteins

The following report details the development of novel methods for characterizing proteins. In one case, a proteolytic microreactor column (PMC) was used to obtain a protein digest for peptide mapping by capillary electrophoresis (CE). A second project used CE to examine the isoform composition of prostate specific antigen (PSA). In addition, an off-line interface was designed to couple CE to a Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) to obtain masses of separated species. The development of these methods will reduce the difficulties often encountered in measuring and characterizing proteins. In the first instance, a PMC was created by immobilizing trypsin onto an epoxy polymeric monolith within a fused silica capillary. The PMC was used to digest beta-casein for characterization by peptide mapping. beta-casein was perfused through the PMC, the effluent collected and separated by CE with detection by UV absorbance. The isoform composition of four preparations of PSA from different venders, PSA-ACT complex, and ACT were characterized using CE. The number of isoforms as well as their relative concentrations varied between samples illustrating the utility of this approach in assessing microheterogeneity. In the third project, glycoforms of PSA and human transferrin were analyzed by a combination of CE and off line MALDI-TOF-mass-spectrometry (MALDI-TOF-MS). Fractions collected directly from the CE were analyzed by MALDI-TOF-MS. Rapid off-line MALDI-TOF-MS on complex electropherograms is a powerful tool for characterization of proteins.