Purification Of Gp96-peptide Complexes From Renal Cell Carcinoma Tissue And Analysis Of Peptides Isolated From Gp96 By Mass Spectrometry

Objective: Renal cell carcinoma is a common malignant tumor of urology surgicalsystem, which accounts for approximately 3% of all solid tumors. At present, radicalnephrectomy is still the most effective therapeutic style for RCC. The contribution toimprove postoperation survival rate of conventional adjuvant therapy is limit.Metastatic renal cell carcinoma remains to be a disease, which is difficult to treatmedically. Immunotherapy is a form of cancer treatment that enhances its scientificpromise and legitimacy with each passing year. Hsp-based cancer vaccine is theresearch focus of immunotherapy. Immunization with autologous gp96 preparationsisolated from cancer cells can elicit a cancer-specific protective T cell immuneresponse that is recallable, which is a prerequisite for gp96 as a therapeutic vaccineagainst cancers. It functions as molecular chaperone and can associate with a varietyof antigenic peptides noncovalently. The collection of gp96-associated peptidesrepresents the antigenic peptide pool of a cell. Since the repertoire of antigenicpeptides in the cell is different between normal and tumor calls and is also distinctamong tumors of the same histology due to the randomness of tumor-specificmutations, a purified sample of gp96-peptide complexes is designed to accommodatethe unique genetic mutations underlying each patient's cancer. This novel personalizedcancer vaccine has made great progress in recent years, but the major challenges arethe limited origination of tissue and the complicated purification procedures, whichmake difficult to bring it to commercial reality. There is an important feature that theantigenicity derived from not gp96 but gp96-associated peptides. So we must furtheranalyze and identify gp96-asssociated peptides, which can provide significantinformation for developing effective cancer vaccine. We extract and purify gp96-peptide complexes from RCC tissue, then analyze the amino acid sequence ofpeptides isolated from gp96 by mass spectrometry. This work can provide rationalefor developing peptide vaccine and assembling gp96-peptide complexes in vitro.Method: In the first part, we collect resected RCC samples. Gp96-peptide complexeswere purified by salting out of ammonium sulfate, Concanavalin-A sepharose affinitychromatography and DEAE-sepharose ion exchange chromatography. Gp96 wasconfirmed by SDS-PAGE and western-blot using gp96 monoclonal antibody. Theconcentration was measured by the means of BCA.In the second part, we analyze the masses of peptides stripped from gp96 byMALDI-TOF mass spectrometry using 4-hydroxy-cyanocinnamic acid as matrix. Thepeptides are sequenced by Q-TOF2. The amino acid sequences are deduced with thesoft of MASCOT.Result: In the first part, (1)gp96 is obtained through this procedure and shown to begp96 as confirmed by western-blot using gp96 monoclonal antibody. (2)20-50μggp96-peptide complexes is obtained from 1g RCC tissue.In the second part, (1) the mass range of peptide associated with gp96 is1046.48-3501.56Da (2)the sequences of two gp96-associated peptides areLVPLEGWGGNVM and PPVYYVPYVVL separately (3)the original protein of twopeptides can not be found.Conclusion: (1)gp96-peptide complexes can be obtained from RCC tissue throughsalting out of ammonium sulfate, affinity chromatography on Con-A sepharose andDEAE-sepharose ion exchange chromatography. (2) gp96-associated peptides aresmall molecular peptides, two peptides are deduced as tumor antigenic peptides.Further analysis must be accomplished before the application in peptide vaccine.