Purification Of Gp96-peptide Complexes From Bladder Cancer Tissue And Analysis Of Peptides Isolated From Gp96 By Mass Spectrometry

Objective: Bladder cancer is a common malignant tumor of urology. Early diagnosis, operation and irrigation of bladder have made great progress, but metastatic and recurrent bladder cancer remains to be a disease which is difficult to treat medically. Many efforts are made to explore comprehensive therapy measures directed to reduce metastasis and recurrence. Immunotherapy is a form of cancer treatment that enhances its scientific promise and legitimacy with each passing year. As a result, this topic has become one of the most highly attended and anticipated of all sessions. Therefore, exploring a promising and effective bladder cancer vaccine seems imperative under the situation. At the extreme of tailoring medicines to those patients who will receive the greatest benefit, is an autologous, or patient specific approach. gp96-peptide complexes purified from tumor is a true custom-based vaccine, which has been shown to induce protective T-cell immunity against a variety of tumors in animal models. This novel personalized cancer vaccine has made great progress in recent years, but the major challenges are the limited origination of tissue and the complicated purification procedures, which make difficult to bring it to commercial reality. There is an important feature, born out by decades of experiment, that the antigenicity derived from not gp96 but gp96-associated peptides. So we must further analyze and identify gp96-asssociatedpeptides, which can provide significant information for developing effective cancervaccine. We extract and purify gp96-peptide complexes from bladder cancer tissue,then analyze the amino acid sequence of peptides isolated from gp96 by massspectrometry. This work can provide rationale for developing peptide vaccine andassembling gp96-peptide complexes in vitro.Method: In the first part, we collect ten resected bladder cancersamples.gp96-peptide complexes was purified by salting out of ammonium sulfate,affinity chromatography on concanavalin-A sepharose and DEAE-sepharose ionexchange chromatography.gp96 was confirmed by SDS-PAGE and western-blotusing gp96 monoclonal antibody. The concentration was measured by the means ofBCA.In the second part, we analyze the masses of peptides isolated from gp96 byMALDI-TOF( matrix-assisted laser desorption/ionization time of flight) massspectrometry using 4-hydroxy-cyanocinnamic acid as matrix. The peptides aresequenced by Q-TOF2.The amino acid sequences are deduced with the softhttp://www.expasy.org/tools/blast/.Result: In the first part, (1) gp96 is obtained through this procedure and shown tobe gp96 as confirmed by western-blot using gp96 monoclonal antibody. (2) 20-40ug gp96-peptide complexes is obtained from 1g bladder cancer tissue.In the second part, (1) the mass range of peptide associated with gp96 is 860.86-3473.05Da ( 2 ) the sequences of two gp96-associated peptides are RFQTWPYVVand RFQTWPYVVL separately (3 ) the original protein of two peptides can not be found. Conclusion: (1) gp96-peptide complexes can be obtained from bladder cancertissue through salting out of ammonium sulfate, affinity chromatography on concanavalin-A sepharose and DEAE-sepharose ion exchange chromatography.(2)gp96-associated peptides are small molecular peptides, two peptides are deduced as tumor antigenic peptides. Further analysis must be accomplished before the application in peptide vaccine.