Developing Mass Spectrometry-Based Analytical Methodologies for Analyzing Complex Protein and Lipid Samples
Creators: name: Hou, Weimin
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2013
Subjects: Proteomics
Lipidomics
protein
lipid
identification
quantification
proteomic reactor
glycerophospholipids

description: Mass spectrometry has increasingly become the method of choice for the analysis of complex biological samples, including proteins and lipids. This thesis describes the development of MS-based analytical methodologies for the analysis of complex proteomic and lipidomic samples. Chapter 3 describes the development of microfluidic proteomic reactors, in the formats of SCX reactor, SCX 96-well plate reactor, and SAX reactor, for the enzymatic digestion of complex proteomic samples for subsequent LC-MS/MS analysis. These microfluidic proteomic reactors greatly simplified the enzymatic digestion of complex proteomic samples by combining multiple processing steps, such as rapid extraction and enrichment of proteins. Furthermore, chemical and enzymatic treatments of proteins were all performed in a few nanoliters effective volume, resulting in an increased protein digestion efficacy. After the protein digestion process, the resulting peptides were eluted in buffers that were compatible with HPLC-MS/MS analysis. In chapter 4, a methodology based on nano-HPLC-ESI-MS/MS for the analysis of PAF and LPC lipid species is described. In this method, lipid extracts from biological samples were separated by nano-flow HPLC prior to being introduced into a Q-TRAP 2000 mass spectrometer, where the lipid species of interest were detected using a precursor ion scan at m/z 184. Absolute quantitation of PAF family lipid species were performed with standard addition method, where 5 standard solutions containing 0.2-1 ng each of C16:0, C18:0 PAF and C16:0, C18:0 lyso-PAF were used in the experiment. Further, the spiking of identical amount of non-endogenous C13:0 LPC at time of extraction allow the relative comparisons of other LPC lipid species of interest between different samples. The developed methods were employed to analyze the changes of PAF and LPC lipid species in NGFdifferentiated PC12 cells, in the posterior/entorhinal cortex of AD patients and TgCRND8 transgenic mice, and over the course of 24 hour exposure of human hNT neurons to Aβ42 treatment, respectively, in comparison to controls. iii Chapter 5 describes the development of a novel shotgun lipidomic methodology for the determination of stereospecificity of diacyl glycerophospholipids including glycerophosphatidic acids (PA), glycerophosphoserines (PS), glycerophosphoglycerols (PG), glycerophosphoinositols(PI), and glycerophosphoethanolamines (PE), which can be conventionally ionized under negative ion mode. The stereospecificity of diacyl glycerophospholipids was determined based on the relative abundance of the lyso-form fragment ions, attributed to the neutral loss of fatty acyl moieties. The fragmentation patterns of a variety of diacyl glycerophospholipid standards were first fully examined over a wide range of collision energy. We observed that lyso-form fragment ions corresponding to the neutral loss of fatty acyl moieties attached to the sn2 position as free fatty acids ([M-Sn2]-) and as ketenes ([M-(Sn2-H2O)]-) exhibited consistently higher intensity than their counter part ions due to the neutral loss of fatty acyl moieties attached to the sn1 position ([M-Sn1]- and [M-(Sn1-H2O)]-). We then examined the product ion spectra of diacyl glycerophospholipids recorded from lipid extracts of rat hepatoma cells, where the stereospecific information of these lipids was conclusively determined.