SRM Assay to Indicate Cancer Therapy

Abstract

The current disclosure provides for specific peptides, and derived ionization characteristics of the peptides, from the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins that are particularly advantageous for quantifying the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins directly in biological samples that have been fixed in formalin by the methods of Selected Reaction Monitoring (SRM) mass spectrometry, or as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed wherein the biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from the biological sample using the Liquid Tissue™ reagents and protocol and the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins are quantitated in the Liquid Tissue™ sample by the method of SRM/MRM mass spectrometry, by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of an ALK, Ros, Ron, Ret, TS, and/or FGFR1 fragment peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

1 . A method for measuring the level of at least one of the human Ros, Ron, Ret, TS, and/or FGFR1 proteins in a human biological sample of formalin-fixed tissue, comprising detecting and quantifying using mass spectrometry the amount of Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptide in a protein digest prepared from said human biological sample; and calculating the level of Ros, Ron, Ret, TS, and/or FGFR1 protein in said sample;
 wherein said amount is a relative amount or an absolute amount.   
     
     
         2 . The method of  claim 1 , further comprising the step of fractionating said protein digest prior to detecting and quantifying the amount of said Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides. 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein said protein digest comprises a protease digest. 
     
     
         5 . The method of  claim 1 , wherein said mass spectrometry comprises tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, and/or time of flight mass spectrometry. 
     
     
         6 . The method of  claim 1 , wherein the Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides comprises an amino acid sequence as set forth as SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16. SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39. 
     
     
         7 - 8 . (canceled) 
     
     
         9 . The method of  claim 1  wherein quantifying the Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides comprises comparing an amount of one or more Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides in one biological sample to the amount of the same ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides in a different and separate biological sample. 
     
     
         10 . The method of  claim 1 , wherein quantifying one or more Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides comprises determining the amount of each of the Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides in a biological sample by comparison to an added internal standard peptide of known amount, wherein each of the Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides in the biological sample is compared to an internal standard peptide having the same amino acid sequence. 
     
     
         11 . The method of  claim 10 , wherein the internal standard peptide is an isotopically labeled peptide. 
     
     
         12 . The method of  claim 1 , wherein detecting and quantifying the amount of one or more Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides in the protein digest indicates the presence of Ros, Ron, Ret, TS, and/or FGFR1 protein and an association with cancer, including lung cancer, in a patient or subject. 
     
     
         13 . The method of  claim 12 , further comprising correlating the results of said detecting and quantifying the amount of one or more Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides, or the amount of said Ros, Ron, Ret, TS, and/or FGFR1 proteins to the diagnostic stage/grade/status of the cancer, including lung cancer. 
     
     
         14 . The method of  claim 13 , wherein correlating the results of said detecting and quantifying the amount of one or more Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides, or the amount of said Ros, Ron, Ret, TS, and/or FGFR1 proteins to the diagnostic stage/grade/status of the cancer is combined with detecting and/or quantifying the amount of other proteins or peptides from other proteins in a multiplex format to provide additional information about the diagnostic stage/grade/status of the cancer, including lung cancer. 
     
     
         15 . The method of  claim 13 , further comprising administering to a patient or subject from which said biological sample was obtained a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent and/or amount of the therapeutic agent administered is based upon the amount of one or more Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides or the amount of Ros, Ron, Ret, TS, and/or FGFR1 proteins. 
     
     
         16 . The method of  claim 15 , wherein the treatment or the therapeutic agent is directed to cancer cells expressing Ros, Ron, Ret, TS, and/or FGFR1 proteins. 
     
     
         17 . (canceled)