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doi:10.1016/j.chroma.2007.03.096 
Copyright © 2007 Elsevier B.V. All rights reserved.
Characterization of apolipoprotein and apolipoprotein precursors in pancreatic cancer serum samples via two-dimensional liquid chromatography and mass spectrometry
Available online 30 March 2007.
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Abstract
Major advances in cancer control depend upon early detection, early diagnosis and efficacious treatment modalities. Current existing markers of pancreatic ductal adenocarcinoma, generally incurable by available treatment modalities, are inadequate for early diagnosis or for distinguishing between pancreatic cancer and chronic pancreatitis. We have used a proteomic approach to identify proteins that are differentially expressed in sera from pancreatic cancer patients, as compared to control. Normal, chronic pancreatitis and pancreatic cancer serum samples were depleted of high molecular weight proteins by acetonitrile precipitation. Each sample was separated by chromatofocusing, and then further resolved by reversed-phase (RP)-HPLC. Effluent from the RP-HPLC column was split into two streams with one directly interfaced to an electrospray time-of-flight (ESI-TOF) mass spectrometer for molecular weight (MW) determination of the intact proteins. The remainder went through a UV detector with the corresponding peaks collected with a fraction collector, subsequently used for MS/MS analysis. The ion intensities of proteins with the same MW obtained from ESI-TOF-MS analysis were compared, with the differentially expressed proteins determined. An 8915 Da protein was found to be up-regulated while a 9422 Da protein was down-regulated in the pancreatic cancer sera. Both proteins were identified by MS and MS/MS as proapolipoprotein C-II and apolipoprotein C-III1, respectively. The MS/MS data of proapolipoprotein C-II was searched using “semi-trypsin” as the search enzyme, thus confirming that the protein at 8915 Da was proapolipoprotein C-II. In order to confirm the identity of the protein at 9422 Da, we initially identified a protein of 8765 Da with a similar mass spectral pattern. Based on MS and MS/MS, its intact molecular weight and “semi-trypsin” database search, the protein at 8765 Da was identified as apolipoprotein C-III0. The MS and MS/MS data of the proteins at 8765 Da and 9422 Da were similar, thus confirming the protein at 9422 Da as being apolipoprotein C-III1. The detection of differentially expressed proapolipoprotein C-II and apolipoprotein C-III1 in the sera of pancreatic cancer patients may have utility for detection of this deadly malignancy.
Keywords: Accurate sequence identification; Serum proteins; Apolipoprotein C-II; Apolipoprotein C-III; Pancreatic cancer; Glycoprotein
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. High molecular weight protein depletion of sera
- 2.2. Serum
- 2.3. Chromatofocusing separation of depleted serum
- 2.4. Determination of differentially expressed proteins with NPS RP-HPLC coupled to ESI-TOF-MS
- 2.5. Trypsin digestion and MALDI-QIT-TOF-MS and MS/MS identification
- 3. Results and discussion
- 3.1. Determination of differentially expressed low molecular weight serum proteins
- 3.2. Identification of proapolipoprotein C-II and mature apolipoprotein C-II
- 3.3. Identification of apolipoprotein C-III0 and apolipoprotein C-III1
- 4. Concluding remarks
- Acknowledgements
- References
