| The following three projects describe the identification of biologically relevant peptides using liquid chromatography interfaced to mass spectrometry. Different methods were used to isolate the peptides/proteins of interest. In Chapter 1, a DNA affinity column was used to isolate DNA-binding proteins. Chapter 2 describes the use of immunopurification to select peptides specific for a certain type of MHC molecule. In Chapter 3, gel electrophoresis was used to isolate proteins from mosquito salivary glands that were potential receptors for a malaria-causing parasite. Identification of these peptides and proteins was greatly facilitated by the use of microcapillary HPLC columns with nanoliter per minute (∼5–200 nL/min) flow rate.; Isolated peptides, either from the surface of cells or products of proteolytic digests, were eluted from a nanoflow HPLC-μESI microcapillary column into an LCQ quadrupole ion trap mass spectrometer (Finnigan, San Jose, CA). The nanoflow HPLC columns allow for increased analyte ion transmission into the mass spectrometer.; Innovations in mass spectrometry, including data-dependent software provided with the LCQ quadrupole ion trap mass spectrometer, enhanced the amount of structural peptide information obtained by limiting the amount of redundant MS/MS spectra acquired and enabling lower abundance peptides to be selected for MS/MS analysis. MS/MS spectra were searched against databases of theoretical peptide spectra from known proteins using SEQUEST. Success from this approach is described in Chapters 1 and 2, where a transcriptional regulatory factor was identified from C. elegans and a novel minor antigen was identified and traced back to a gene on the human Y chromosome, respectively. Chapter 3 describes the identification of one potential protein receptor for Plasmodium gallinaceum and relates the problems that arose from a lack of information in protein databases and the attempts to work around them. |
