DEVELOPMENT AND OPTIMIZATION OF ON-PROBE AFFINTY CAPTURE (OPAC) MALDI MASS SPECTROMETRY FOR THE FRACTIONATION AND ANALYSIS OF COMPLEX PROTEIN MIXTURES

• Main Author: Fernando, Ganga Sripali
• Format: Others
• Published: OpenSIUC 2009
• Subjects: MALDI; Mass Spectrometry; Plasma polymers; Protein separation; Proteomics; RF pulsed plasma
• Online Access: https://opensiuc.lib.siu.edu/dissertations/68; https://opensiuc.lib.siu.edu/cgi/viewcontent.cgi?article=1068&context=dissertations

A high throughput proteomic analysis method is described here that uses more economically favorable, easily manufactured probe surface that can be directly incorporated on the MALDI target. On-Probe Affinity Capture (OPAC) MALDI is a method that uses the RF pulsed plasma modified target surfaces for the protein purification, separation and identification all on the same single probe and one of the highest advantage of this method is the number of different experiments that can be carried out simultaneously using the intelligent design of the probe. The new design of the OPAC probe presented in this dissertation gives the ability to perform about 100 different experiments on one single MALDI target. These OPAC probes can be used for the fractionation and analysis of proteins from complex biologically derived samples. The separated proteins can be identified on the OPAC probe using it directly as the MALDI target and selecting a proper elution solution that depends on the chemistry of the OPAC probe, the surface bound proteins can be eluted and incorporated into the matrix crystal. This dissertation focuses mainly on developing this method for analysis of different samples. A tryptic digest of a single protein was separated and identified by submitting the peak lists to MASCOT database search and the sequence coverage obtained before and after fractionation has been compared. Then a mixture of tryptic peptides of five different known proteins were fractionated on OPAC surfaces and the identification of proteins obtained was compared before and after fractionation. Further developing this technique, biologically derived mixtures of proteins from two different well studied sources have been analyzed using OPAC-MALDI. Escherichia coli bacterial proteome was digested and fractionated and the peptides were studied using De Novo sequencing method and their affinity fractionation behavior is confirmed by calculating the iso electric point (pI) and the hydrophobicity of the predicted peptide sequence. Synechosystis sp PCC 6803 was cultured and the protein extracts were prepared for the OPAC studies. The clear fractionated of this mixture was observed and the amount of information derived after fractionation is found to be significantly higher than the unfractionated sample. Taking a slightly different approach, a phosphoprotein binding OPAC probe was prepared using commercially available poly(methyl methacrylate) (PMMA) film. The hydrolyzed PPMA films were reacted with CuCl2 solution to incorporate metal ions on the surface by electrostatic interaction, which then facilitates the phosphoprotein binding on the OPAC probe. This was demonstrated using a binary mixture of commercially available peptides and fractionating the mixture on Cu-impregnated PMMA film. Finally, in a collaborative work, the possibility of increased surface binding capacity was explored by using a synthetic organic nanosponge surface that expands and collapses due to change of pH. These brush polymers were prepared by Dyer group and a binary mixture of peptides were fractionated and analyzed by MALDI MS.