Analysis of human serum protein extracts by matrix assisted laser desorption ionization and electrospray ionization quadrupole-quadrupole time-of-flight tandem mass spectrometry

Serum is a complex heterogeneous matrix contianing endogenous proteins with potential diagnostic value. No previously reported methods have allowed the detection of all its constituent proteins from a single sample. In the work described in this thesis, normal human serum was separated by reversed-phase or dye affinity chromatography, ultrafiltration and by extraction under various pH and salt concentrations in aqueous and organic solutions. The serum extracts were analyzed by SDS-PAGE, MALDI and LC-ESI-Qq-TOF MS/MS without prior enzymatic digestion. Peptides observed as multiple isotopic peaks in MS mode were correlated with known serum proteins by ProteinPilot and X!Tandem analysis of their fragmentation spectra using the no enzyme [XX] condition. Fragment ion masses within 0.04 Da of those predicted yielded high confidence identifications from both algorithms. Angiotensin 1 and [G1u1] fibrinopeptide B were clearly detected in serum spiked with as little as 250 and 100 femtomoles respectively. Post-translational modifications of serum peptides were identified using ProteinPilot.

Analysis of human serum protein extracts by matrix assisted laser desorption ionization and electrospray ionization quadrupole-quadrupole time-of-flight tandem mass spectrometry

Serum is a complex heterogeneous matrix contianing endogenous proteins with potential diagnostic value. No previously reported methods have allowed the detection of all its constituent proteins from a single sample. In the work described in this thesis, normal human serum was separated by reversed-phase or dye affinity chromatography, ultrafiltration and by extraction under various pH and salt concentrations in aqueous and organic solutions. The serum extracts were analyzed by SDS-PAGE, MALDI and LC-ESI-Qq-TOF MS/MS without prior enzymatic digestion. Peptides observed as multiple isotopic peaks in MS mode were correlated with known serum proteins by ProteinPilot and X!Tandem analysis of their fragmentation spectra using the no enzyme [XX] condition. Fragment ion masses within 0.04 Da of those predicted yielded high confidence identifications from both algorithms. Angiotensin 1 and [G1u1] fibrinopeptide B were clearly detected in serum spiked with as little as 250 and 100 femtomoles respectively. Post-translational modifications of serum peptides were identified using ProteinPilot.

Oxidation in Low Moisture Foods as a Function of Surface Lipids and Fat Content

Lipid oxidation is a major limitation to the shelf-life of low moisture foods and can lead to food waste. Little is known of whether the surface lipids in low moisture foods are more susceptible to oxidation since they are exposed to the environment. Therefore, the purpose of this research is to compare the rate of oxidation in surface and total lipids. Lipids in crackers were found to be in a heterogeneous matrix with proteins and starch, as determined by confocal microscopy. However, unlike spray-dried powders, both surface and interior lipids oxidized at similar rates, suggesting that the cracker matrix was not able to protect lipids from oxidation. Increasing the fat content of the crackers increased oxidation rates, which could be due to differences in the lipid structure or higher water activities in the high-fat crackers.