Development of Mass Spectrometric Methods for Membrane Proteome Analysis and Protein Sequence Mapping

• Author / Creator

  Sun, Difei

• This thesis work mainly focused on the development of mass spectrometric methods for membrane proteome analysis and protein sequence mapping. First, the comparison of the performance of RapiGest-, PPS- and SDS-based sample preparation methods for shotgun membrane proteome analysis was investigated. We demonstrated that the use of RapiGest allows identification of more peptides and proteins than the use of PPS or SDS. Second, based on the RapiGest-assisted membrane protein analysis method, a high throughput plasma membrane protein identification and quantitation strategy was developed for the analysis of ALK+ ALCL cell lines. Using this method, 561 and 552 unique plasma membrane proteins and extracellular proteins were identified from Karpas 299 cell line and SUPM2 cell line, respectively; 48 unique plasma membrane proteins and extracellular proteins were found to be differentially expressed between NPM-ALK expressing HEK 293 cells and NPM-ALK absent HEK 293 cells by quantification analysis; and six putative biomarkers were chosen for further biological validation. Third, an integrated strong-cation exchange liquid chromatographic procedure for SDS removal and peptide separation for SDS-assisted shotgun proteome analysis was developed, which allowed effective SDS removal while keeping a high peptide recovery rate when the low-cost SDS-assisted sample preparation method was used. We have shown that the performance of this method is better than that of the RapiGest-assisted method. Fourth, a protein sequence mapping method was developed based on the use of gel electrophoresis to separate proteins, followed by in-gel MAAH of the gel-separated proteins using TFA to produce peptides and LC-MS/MS of the resultant peptides. This method provided high sequence coverage of proteins separated from complex protein mixture. To demonstrate the applications of this method, 19 relatively high abundance human plasma proteins were mapped with high sequence coverage. In addition, bovine alpha-S1-casein phospho-isoforms were characterized and six new phosphorylation sites were identified. The methods developed in this thesis work have been shown to be useful in proteomics research. The membrane proteome analysis methods are applicable to study any cell lines or tissues and the protein sequence mapping method can be applied to characterize a variety of proteins with virtually no molecular weight limit.

• Subjects / Keywords

  • Protein sequence mapping
  • Mass-spectrometry
  • Membrane proteome

• Graduation date

  Spring 2014

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3SJ1B26T

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.