The development of separations and mass spectrometry technologies for the analysis of biomolecules.

Abstract

The goal of this work was to develop liquid phase separations and mass spectrometric detection methods for the analysis of DNA and proteins. The methods are intended to replace gel electrophoresis in order to improve the speed, reproducibility and resolving power for molecular biological procedures used in genomic and proteomic studies. Four projects were undertaken which illustrate different aspects of this approach. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) has been applied to the detection of DNA restriction fragments for a common diagnostic test for apolipoprotein E, a risk factor for Alzheimer's disease. A reversed-phase HPLC method for the separation of DNA restriction fragments and Polymerase Chain Reaction (PCR) products was also developed. Compared with gel electrophoresis, both methods reduce analysis times from several hours to a few minutes or less. A two-dimensional (2-D) liquid phase separation technique that separates proteins according to isoelectric point (pI) in the first dimension and hydrophobicity using reversed phase HPLC in the second dimension was developed as an alternative to 2-Dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE). The liquid phase technique produces a map of cellular proteins similar to that produced by 2-D PAGE where the proteins in the liquid phase after separation, thus simplifying further analysis. This method requires 8 hours to complete, compared with 3 days to complete a 2-D PAGE gel, and is highly reproducible. A three-dimensional separation technique was developed where the same 2-D liquid phase separation (pl vs. hydrophobicity) is used, after which the proteins are injected on-line into an Electrospray Ionization Time of Flight Mass Spectrometer (ESI-TOF MS) that gives highly accurate masses for the intact proteins. The use of three parameters (pI, hydrophobicity and protein mass) as a basis for comparison of proteins expressed in cells under different conditions (i.e normal vs. diseased) ensures that the majority of common proteins can be confidently identified, allowing researchers to quickly assess the presence of biologically significant changes in proteins expressed in different cell types. This method can also reveal post-translational modifications (PTMs), because mass shifts due to PTMs are much larger than the variance in protein masses detected by this method.