The development of Proteosep and protein microarrays as well as their applications to cancer proteomics with mass spectrometric methods.

Abstract

This work develops a multidimensional liquid-phase separation protocol---Proteosep technique---for the proteome fractionation of whole-cell lysates. The 1<super>st</super> dimension fractionates proteins by pI using chromatofocussing with analytical columns. The 2<super>nd</super> dimension separates the proteins in each pI fraction using nonporous reversed-phase (NPS-RP) HPLC. A 2D map of each cell line (pI vs. hydrophobicity) as detected by UV absorption is generated with Proteovue(TM) and Deltavue(TM) software. The separated proteins are analyzed by mass spectrometry to determine protein MW and IDs. One of the important applications of this technique compares the protein expression profiles between drug-treated and untreated colon cancer HCT-116 cells. In the 2D UV map produced after the LC fractionations, over 1000 protein bands can be detected in 0.2 pH fractions over pH4--7. A differential display map indicating the presence of up- or down-regulated proteins is also generated. The liquid eluent from the separation is directed on-line into an electrospray (ESI) TOF-MS to obtain accurate MWs of the intact proteins. These MWs together with the pI values and a peptide map are then used to identify proteins by database searching. Proteins up- or down-regulated as a result of the drug treatment are identified and the quantitation of the change in the protein expression is based upon UV absorption. The method has been shown to provide efficient separations and is highly reproducible for quantitative analyses of lysates via differential display mapping. This work also performs proteomic analyses in a high throughput fashion using protein microarrays to identify potential serum biomarkers for prostate cancer. The cell lysate of prostate cancer is fractionated in a 2D LC separation and arrayed on nitrocellulose slides. Specific protein fractions are immunoreactive against prostate cancer serum but not against serum from healthy individuals. The molecular mass and peptide sequence data obtained in the mass spectrometric analysis of these fractions identify 3 protein antigens. These proteins might serve as sero-diagnostic markers for prostate cancer. Importantly, this method uses post-translationally modified proteins as baits for the detection of humoral response, and is amenable to automation due to the fractionation of proteins in the liquid phase.