Methods of isolating barrel-like proteases and identifying peptides processed thereby

Abstract

A method of isolating barrel-like proteases is disclosed. The method comprising isolating barrel-like proteases from a biological sample containing the barrel-like proteases under conditions that maintain the content of the barrel-like protease-processed peptides in the barrel-like proteases upon isolation. A method of isolating barrel-like protease-processed peptides and a method of identifying barrel-like protease-processed peptides are also disclosed.

What is claimed is: 
     
         1 . A method of isolating barrel-like proteases, the method comprising isolating barrel-like proteases from a biological sample containing said barrel-like proteases under conditions that maintain the content of the barrel-like protease-processed peptides in said barrel-like proteases upon said isolation, thereby isolating the barrel-like proteases, wherein said conditions that maintain the content of the barrel-like protease-processed peptides in said barrel-like proteases comprise a cross-linking agent and wherein said barrel-like protease -processed peptides are non-tryptic peptides. 
     
     
         2 . A method of isolating barrel-like protease-processed peptides, the method comprising:
 (a) isolating barrel-like proteases according to  claim 1 ; and   (b) isolating a peptide pool from total proteinaceous pool of said isolated barrel-like proteases, thereby isolating the barrel-like protease -processed peptides, wherein said conditions that maintain the content of the barrel-like protease-processed peptides in said barrel-like proteases comprise a cross-linking agent and wherein said barrel-like protease -processed peptides are non-tryptic peptides.   
     
     
         3 . A method of identifying barrel-like protease-processed peptides, the method comprising:
 (a) isolating the barrel-like protease-processed peptides according to  claim 2 ; and   (b) subjecting the barrel-like protease-processed peptides to sequence analysis, thereby identifying the barrel-like protease-processed peptides.   
     
     
         4 . The method of any one of  claim 2 , wherein said conditions that maintain the content of the barrel-like protease-processed peptides in said barrel-like proteases comprise contacting the barrel-like proteases with a peptidase inhibitor. 
     
     
         5 . The method of  claim 2 , wherein said barrel-like protease is a proteasome. 
     
     
         6 . The method of  claim 2 , wherein said biological sample is from a healthy tissue or cell, malignant tissue or cell, an inflamed tissue or cell, a senescent tissue or cell, an immature tissue or cell, a senescent tissue or cell, or a pathogen infected tissue or cell. 
     
     
         7 . The method of  claim 2 , wherein said biological sample is selected from the group consisting of intact cells in suspension, tissue biopsy, cell-free proteasomes, exosomes and microparticles. 
     
     
         8 . The method of  claim 2 , wherein said cell sample comprises a body fluid, a cell line or a primary cell. 
     
     
         9 . The method of  claim 2 , wherein said body fluid is selected from the group consisting of whole blood, fractionated blood, serum, plasma, cerebrospinal fluid, urine, lymph fluid, an external secretion of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, peritoneal lavage and amniotic fluid. 
     
     
         10 . The method of  claim 2 , wherein said biological sample comprises immune cells, cancer cells, stem cells, progenitor cells, senescent cells or pathogen-infected cells. 
     
     
         11 . The method of  claim 2 , wherein said isolating said barrel-like proteases is effected following cell lysis. 
     
     
         12 . The method of  claim 2 , wherein said isolating said barrel-like proteases is effected by immunoprecipitation using an antibody to said barrel-like protease or a protein associated therewith. 
     
     
         13 . The method of  claim 2 , wherein said isolating a peptide pool from total proteinaceous pool of said isolated barrel-like proteases is effected by solvent precipitation followed by solid phase extraction. 
     
     
         14 . A barrel-like protease preparation obtainable according to the method of any one of  claim 1 . 
     
     
         15 . A peptide preparation obtainable according to the method of  claim 2 . 
     
     
         16 . A computer readable storage medium comprising an annotated sequence data of the peptide preparation of  claim 15 . 
     
     
         17 . A method of testing an effect of a test compound or condition, the method comprising:
 (a) contacting a biological sample comprising barrel-like proteases with the test compound; and subsequently   (b) identifying barrel-like protease processed peptides according to  claim 3 .