csPCNA Isoform Modifications And Uses Thereof

Abstract

Methods and compositions to detect the presence of csPCNA isoform by identifying one or more posttranslational modifications are disclosed. Methods to identify csPCNA isoform through posttranslational modifications including methylesterification levels are disclosed.

1 . A method of diagnosing or prognosing malignancy, the method comprising:
 detecting a post-translational modification comprising a methyl ester on one or more of 16 aspartic acid or glutamic acid residues corresponding to the amino acid positions 3, 85, 93, 94, 104, 109, 115, 120, 132, 143, 174, 189, 201, 238, 256, and 259 of SEQ ID NO: 30 in a biological sample suspected of containing a csPCNA isoform; comparing the levels of methyl esters on the csPCNA isoform with a nonmalignant isoform of PCNA, wherein the csPCNA isoform has a lower level of methyl esterification than the nonmalignant isoform of PCNA; and   diagnosing or prognosing malignancy based on detection of csPCNA in the biological sample.   
     
     
         2 . The method of  claim 1 , wherein the biological sample is a bodily fluid. 
     
     
         3 . The method of  claim 2 , wherein the bodily fluid is selected from blood, plasma, lymph, serum, pleural fluid, spinal fluid, saliva, sputum, urine, gastric juice, pancreatic juice, ascites fluid, synovial fluid, milk, and semen. 
     
     
         4 . The method of  claim 1 , wherein the biological sample is a tissue sample. 
     
     
         5 . The method of  claim 4 , wherein the tissue is selected from breast, prostate, lung, colon, epithelial, connective, cervical, esophageal, brain, thymus, thyroid, pancreas, testis, ovary, intestine, bladder, stomach, soft tissue sarcomas, osteosarcoma, leukemia, lymphoma, carcinoma, adenocarcinoma, placenta, fibrous, germ cell tissue, and extracts thereof. 
     
     
         6 . The method of  claim 1 , wherein the methyl ester present on one or more of the 16 aspartic acid or glutamic acid residues corresponds to the peptides with modified amino acid residues selected from 
       
         
           
                 
                 
                 
               
                     
                   MFE m AR; 
                   (SEQ ID NO: 1) 
                 
                     
                     
                 
                     
                   IE m DEEGS; 
                   (SEQ ID NO: 2) 
                 
                     
                     
                 
                     
                   IEDEE m GS; 
                   (SEQ ID NO: 3) 
                 
                     
                     
                 
                     
                   VSDYE m MK; 
                   (SEQ ID NO: 4) 
                 
                     
                     
                 
                     
                   MPSGE m FAR; 
                   (SEQ ID NO: 5) 
                 
                     
                     
                 
                     
                   LSQTSNVD m K; 
                   (SEQ ID NO: 6) 
                 
                     
                     
                 
                     
                   CAGNE m DIITLR; 
                   (SEQ ID NO: 7) 
                 
                     
                     
                 
                     
                   FSASGE m LGNGNIK; 
                   (SEQ ID NO: 8) 
                 
                     
                     
                 
                     
                   AEDNADTLALVFEAPNQE m K; 
                   (SEQ ID NO: 9) 
                 
                     
                     
                 
                     
                   AE m DNADTLALVFEAPNQEK; 
                   (SEQ ID NO: 10) 
                 
                     
                     
                 
                     
                   AED m NADTLALVFEAPNQEK; 
                   (SEQ ID NO: 11) 
                 
                     
                     
                 
                     
                   AEDNADTLALVFE m APNQEK; 
                   (SEQ ID NO: 12) 
                 
                     
                     
                 
                     
                   LMD m LDVEQLGIPEQEYSCVVK; 
                   (SEQ ID NO: 13) 
                 
                     
                     
                 
                     
                   ATPLSSTVTLSMSADVPLVVE m YK; 
                   (SEQ ID NO: 14) 
                 
                     
                     
                 
                     
                   LSQTSNVDKEEEAVTIEMNE m PVQLTFALR; 
                   (SEQ ID NO: 15) 
                 
                 
                 
               
                     
                   and 
                 
                     
                     
                 
                 
                 
                 
               
                     
                   LMDLDVEQLGIPEQE m YSCVVK, 
                   (SEQ ID NO: 31) 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
             
                
                
               
            
             
                
               
            
           
         
         wherein E m  represents a methylesterified glutamic acid residue and D m  represents a methylesterified aspartic acid residue. 
       
     
     
         7 . The method of  claim 1 , wherein the detection of csPCNA isoform is performed using a mass spectrometric analysis. 
     
     
         8 . The method of  claim 7 , wherein the mass spectrometric analysis is liquid chromatography (LC) mass spectrometric (MS) analysis. 
     
     
         9 . The method of  claim 7 , wherein the mass spectrometric analysis of a csPCNA-derived peptide results in a 14 Da mass shift as compared to a corresponding unmodified peptide. 
     
     
         10 . A modified proliferating cell nuclear antigen (PCNA) peptide comprising an amino acid sequence selected from the group consisting of: 
       
         
           
                 
                 
                 
               
                     
                   MFE m AR; 
                   (SEQ ID NO: 1) 
                 
                     
                     
                 
                     
                   IE m DEEGS; 
                   (SEQ ID NO: 2) 
                 
                     
                     
                 
                     
                   IEDEE m GS; 
                   (SEQ ID NO: 3) 
                 
                     
                     
                 
                     
                   VSDYE m MK; 
                   (SEQ ID NO: 4) 
                 
                     
                     
                 
                     
                   MPSGE m FAR; 
                   (SEQ ID NO: 5) 
                 
                     
                     
                 
                     
                   LSQTSNVD m K; 
                   (SEQ ID NO: 6) 
                 
                     
                     
                 
                     
                   CAGNE m DIITLR; 
                   (SEQ ID NO: 7) 
                 
                     
                     
                 
                     
                   FSASGE m LGNGNIK; 
                   (SEQ ID NO: 8) 
                 
                     
                     
                 
                     
                   AEDNADTLALVFEAPNQE m K; 
                   (SEQ ID NO: 9) 
                 
                     
                     
                 
                     
                   AE m DNADTLALVFEAPNQEK; 
                   (SEQ ID NO: 10) 
                 
                     
                     
                 
                     
                   AED m NADTLALVFEAPNQEK; 
                   (SEQ ID NO: 11) 
                 
                     
                     
                 
                     
                   AEDNADTLALVFE m APNQEK; 
                   (SEQ ID NO: 12) 
                 
                     
                     
                 
                     
                   LMD m LDVEQLGIPEQEYSCVVK; 
                   (SEQ ID NO: 13) 
                 
                     
                     
                 
                     
                   ATPLSSTVTLSMSADVPLVVE m YK; 
                   (SEQ ID NO: 14) 
                 
                     
                     
                 
                     
                   LSQTSNVDKEEEAVTIEMNE m PVQLTFALR; 
                   (SEQ ID NO: 15) 
                 
                 
                 
               
                     
                   and 
                 
                     
                     
                 
                 
                 
                 
               
                     
                   LMDLDVEQLGIPEQE m YSCVVK; 
                   (SEQ ID NO: 31) 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
             
                
                
               
            
             
                
               
            
           
         
         wherein E m  represents a methylesterified glutamic acid residue and D m  represents a methylesterified aspartic acid residue. 
       
     
     
         11 . The modified peptide of  claim 10 , wherein the peptides are post-translationally modified. 
     
     
         12 . The modified peptide of  claim 10 , wherein the peptides are exposed to a protease digestion step. 
     
     
         13 . The modified peptide of  claim 10 , wherein the peptides are synthetic.