SRM assay to indicate cancer therapy
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 the human ALK protein in a human biological sample of formalin-fixed tissue, comprising detecting and quantifying using mass spectrometry the amount of an ALK protein fragment peptide in a protein digest prepared from said human biological sample; and calculating the level of ALK protein in said sample;
wherein said fragment peptide is the peptide of SEQ ID NO:5, and
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 ALK protein fragment peptides.
3. The method of claim 1 , wherein said protein digest comprises a protease digest.
4. The method of claim 1 , wherein quantifying said ALK protein fragment peptide comprises comparing the amount of said ALK protein fragment peptide in one biological sample to the amount of the same ALK protein fragment peptide in a different and separate biological sample.
5. The method of claim 4 , wherein quantifying said ALK protein fragment peptide comprises determining the amount said ALK protein fragment peptide in a biological sample by comparison to an added internal standard peptide of known amount, wherein the ALK protein fragment peptide in the biological sample is compared to an internal standard peptide having the same amino acid sequence.
6. The method of claim 5 , wherein the internal standard peptide is an isotopically labeled peptide.
7. The method of claim 1 , wherein detecting and quantifying the amount of said ALK protein fragment peptide in the protein digest indicates the presence of ALK protein and an association with cancer in a patient or subject.
8. The method of claim 7 , further comprising correlating the results of said detecting and quantifying the amount of said ALK protein fragment peptide, or the amount of said ALK protein to the diagnostic stage/grade/status of the cancer.
9. The method of claim 8 , wherein correlating the results of said detecting quantifying the amount of said ALK protein fragment peptide, or the amount of said ALK protein 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.
10. The method of claim 8 , 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 said ALK protein fragment or the amount of ALK protein.
11. The method of claim 10 , wherein the treatment or the therapeutic agent is directed to cancer cells expressing ALK protein.
12. The method of claim 7 , wherein said cancer is lung cancer.
13. The method of claim 1 , further comprising detecting and quantifying the amount of a Ros protein fragment peptide in said protein digest.