Characterization of biological compounds by mass spectrometry
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Date
2000-05-01T00:00:00Z
Authors
McComb, Mark Errol
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Abstract
Mass spectrometry is a powerful method for the characterization of biological molecules. However, sample purification is often required due the presence of contaminants within the biological matrix which will affect the quality of spectra. In this thesis, two methods of "on-line" sample purification and separation were explored. Non-porous ether type polyurethane (PU) membranes and porous PU thin films were used as sample supports for matrix assisted laser desorption time-of-flight (MALDI-TOF) MS. Protein and peptide samples were applied directly to the surface of the PU supports. Hydrophobic interactions between the proteins and peptides and the PU membrane allowed the incorporation of a washing step to remove salts and other matrix components present. This provided for an increase in resolution and mass accuracy and thus superior results compared with other supports. Peptide mass mapping was facilitated through on-membrane proteolytic digestion. A procedure for the sampling and off-site analysis of hemoglobin variants was developed by characterizing the Hb Shepherds Bush hemoglobin variant, b74 (E18)Gly [right arrow] Asp A novel application was explored by characterizing water insoluble wheat proteins which consist of gliadins, low molecular weight (LMW) and high molecular weight (HMW) glutenins. 'In vitro' and 'in vivo' qualitative and semi-quantitative analysis of plasma proteins demonstrated the first study of MALDI-TOFMS characterization of multiple proteins adsorbed onto the surface of a model biomaterial (PU). On-line capillary electrophoresis electrospray ionization mass spectrometry (CE/ESI-MS) and tandem mass spectrometry (CE/ESI-MS/MS) experiments were conducted on a Micromass(tm) triple quadrupole mass spectrometer equipped with a Z-Spray(tm) source. On-line CE-time-of-flight mass spectrometry (CE/ESI-TOFMS) experiments were conducted using a reflecting TOF mass spectrometer built in-house. Gold coated sheathless interfaces were used to couple uncoated fused silica capillaries with the mass spectrometers. An electrical system and ESI interfaces were designed and characterized using standard peptides and proteins. For a standard peptide mixtures (10-4 to 10-6 M) (pmol and sub-pmol injection), separation efficiency was typically characterized by N > 104 theoretical plates with S/N ca. 200 to >400. Unit mass resolution (m/[Delta]mFWHM) was obtained on the QQQ and mass accuracy was better than 100 ppm. Reflecting TOFMS afforded mass resolution R > 6000 and mass accuracy of ca. 30 ppm. CE/ESI-MS/MS was used to sequence standard peptides. Selected ion electrophorograms of tryptic peptides of several proteins allowed for peptide mass mapping in spite of poorly resolved total ion electrophorograms. Citrate synthase, SB hemoglobin and equine myoglobin were characterized.