4.7 Article

Characterization of proteins in human pancreatic cancer serum using differential gel electrophoresis and tandem mass spectrometry

期刊

JOURNAL OF PROTEOME RESEARCH
卷 4, 期 5, 页码 1742-1751

出版社

AMER CHEMICAL SOC
DOI: 10.1021/pr050174l

关键词

serum; biomarker; pancreatic cancer; mass spectrometry; albumin-depletion; proteomics; DIGE; immunoaffinity depletion

资金

  1. NCI NIH HHS [R01 CA 95586] Funding Source: Medline
  2. NCRR NIH HHS [S10 RR 019221] Funding Source: Medline
  3. NIDDK NIH HHS [T32 DK 007066, P30 DK 050306] Funding Source: Medline

向作者/读者索取更多资源

The purpose of this study was to develop techniques for identifying cancer biomarkers in human serum using differential in-gel electrophoresis (DIGE), and characterizing the protein biomarkers using tandem mass spectrometry (MS/MS). A major problem in profiling protein expression by DIGE comes from the presence of high concentrations of a small number of proteins. Therefore, serum samples were first chromatographed using an immunoaffinity HPLC column (Agilent Technologies), to selectively remove albumin, immunoglobulins, transferrin, haptoglobin, and antitrypsin. Serum samples from three individuals with pancreatic cancer and three individuals without cancer were compared. Serum samples were processed using the immunoaffinity column. Differential protein analysis was performed using DIGE. A total of 56 protein spot-features were found to be significantly increased and 43 significantly decreased in cancer serum samples. These spot features were excised, trypsin digested, and analyzed by MALDI/TOF/TOF (4700 Proteomics Analyzer, Applied Biosystems). We identified 24 unique proteins that were increased and 17 unique proteins that were decreased in cancer serum samples. Western blot analysis confirmed increased levels of several of these proteins in the pancreatic cancer serum samples. In an independent series of serum samples from 20 patients with pancreatic cancer and 14 controls, increased levels of apolipoprotein E, alpha-1-antichymotrypsin, and inter-alpha-trypsin inhibitor were found to be associated with pancreatic cancer. These results suggest that affinity column enrichment and 2-D DIGE can be used to identify numerous proteins differentially expressed in serum from individuals with pancreatic cancer.

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