| Proteomics is a newly emerging field in the post-genomics era. The development of knowledge and technology in proteomics has provided a new approach to investigate cancer markers. The current biomarkers for malignancy cancers have a poor diagnostic value. Therefore, it is urgent to find the sensitive, specific, and convenient new biomarkers.The separation and identification of proteins in cells or tissues are key problems in proteomics research. Recently, because of high resolving power, reproducibility and automation, multidimensional high-performance liquid chromatography (MDLC) becomes a powerful tool in proteomics research. In this thesis, a new method with an analytical-scale chromatographic cake (10 mm×20 mm I.D.) was used to the fast separation of proteins in human serum, and a platform of off-line multidimensional liquid chromatography coupled with matrix assisted laser desorption ionization time of flight mass Spectrometry (MALDI-TOF MS) was established. And we also studied the biomarker screening of pancreatic cancer serum and tissue by off-line SEC-RPLC-MS. The satisfied results were achieved.The thesis includes following four parts:1. Introduction. A short review on the recent progresses of proteomics, separation technologies, biological mass Spectrometry, the disease proteomics and the new development of pancreatic cancer research was summarized.2. Fast separation and preparation of proteomics samples of human serum with high performance hydrophobic interaction chromatographic cake. A new method of chromatographic cake (10 mm×20 mm I.D.) combined with RPLC off-line was used to fast isolate human serum and enrich low abundance protein in the serum, and detected by MALDI-TOF MS. Four kinds of standard proteins with very low concentration were studied as model proteins to validate this approach. It shows that the MS detection limits of the enriched cytochrome-C and myoglobin can reach to be 1 pmol·μL-1. The presented method was applied to the investigation of human serum proteomics. With increasing of the loading sample volume of human serum on chromatographic cake, the MS signal intensity of detecting protein/peptide become stronger, and more fractions can be detected with MALDI-TOF MS. 285 fractions (<15 kD) can be found when 1.0 mL serum sample was loaded on the chromatographic cake. In addition, the low abundance cytochrome-C as an internal standard also can be separated and enriched successfully with the above method when 1μg cytochrome-C was added into 0.5 mL original serum. The results prove that 2D-LC of the chromatographic cake-RPLC coupled with MALDI-TOF MS can be applied not only to the fast separation and preparation of human serum sample with large loading volume in one cycle of analysis, but also for the efficient isolation and enrichment of the lower abundance proteins/peptides in human serum because of the elimination the effects of the high abundance proteins on the low abundance ones. In addition, the detection efficiency of the low abundance proteins/peptides in human serum with MALDI-TOF MS can also be increased successfully.3. Studied on the biomarker screening of pancreatic cancer serum with size exclusion / reverse phase liquid chromatography - mass Spectrometry (SEC-RPLC-MS). In this part, the serum sample was denatured by 6 mol·L-1 urea solution (1:1, V:V), which can eliminate the interactions between albumin and other proteins/peptides in serum. Then the sample was separated by off-line SEC-RPLC, and all collected fractions were detected by MALDI-TOF MS. The results showed that this method was an excellent platform for serum proteomics because of its good reproducibility and stability. With the comparison of the results obtained from healthy human serum and the serum before a surgical operation and the postoperative sourced from the same individual patient, one differential protein/peptide was found to be the biomarker candidates with m/z of 5734, which was expressed increasingly in the serum of pancreatic cancer patient with sensitivity of 78.38%(29/37) and specificity of 100%(6/6).4. Studied on pancreatic tissue proteomics with SEC-RPLC-MS. The extraction solutions of pancreatic tissue protein were separated by off-line SEC-RPLC, and detected by MALDI-TOF MS. More than 240 proteins/peptides were founded. This method can be applied as a common method in the investigation of tissue proteomics. 11 pairs of cancer pancreas and 3 normal pancreas were analyzed, some proteins/peptides were found to be differently expressed between cancer tissues and normal tissues, and we also found that the protein/peptide with m/z of 5734 was expressed in cancer tissues.
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