Background: Early diagnosis is important for timely treatment of gallbladder carcinoma (GBC) patients leading to the increased survival rate. Here, we have applied serological proteome analysis (SERPA), an immunoproteomics approach, for detection of tumor-associated antigens (TAAs) eliciting humoral response in early stage GBC patients.
Methodology: Immunodepleted tissue proteins from GBC patients (n=7) were resolved by two-dimensional gel electrophoresis (2-DE) followed by immunoblotting using pooled blood plasma from healthy volunteers (n=11) or gallstone (GS) cases (n=11) or early stages of GBC (n=5) or advanced stages of GBC cases (n=9). Image analysis was performed using PDQuest software to identify protein spots with significantly high or specific immunoreactivity in GBC cases. The corresponding protein spots were excised from the 2-D gel followed by in-gel trypsin digestion and mass spectrometric analysis (LC-MS/MS) for identification of proteins. Two of the identified proteins were verified for autoantibody levels in individual plasma samples (30 cases and 20 controls) by dot blot assay.
Findings: 2-D immunoblot analysis led to identification of 25 protein spots showing either significantly high or specific immunoreactivity in early and/or advanced stages of GBC. Mass spectrometric analysis led to identification of proteins from the immunoreactive spots, including annexin A1 (ANXA1), and heat shock protein 60 (HSP60), carbonic anhydrase isoform 1 and 2, aldolase B and cathepsin D. Evaluation of autoantibody levels in individual plasma samples against two of the recombinant proteins, ANXA1 (an immunomodulatory protein implicated in cancer) and HSP60 (chaperonin involved in regulating apoptosis in cancer) using Dot blot assay showed significantly higher levels of autoantibodies against HSP60 (unpaired t-test, p= 0.023) in early stage GBC cases.
Conclusions: The study suggest that the autoantibody levels against HSP60 may be potentially employed for detection of GBC patients at early stages, however, the autoantibody level needs to be validated in larger cohort of clinical samples.