MRM/SRM assay for death receptor 5 protein
Specific peptides, and derived ionization characteristics of those peptides from Death Receptor 5 (DR5) protein are provided that are particularly advantageous for quantifying the DR5 protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring/Multiple Reaction Monitoring (SRM/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 a biological sample using the Liquid Tissue™ reagents and protocol, and the DR5 protein 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 for one or more of the DR5 protein. These peptides can be quantitated if they reside in a modified or in an unmodified form. An example of a modified form of a DR5 peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.
1. A method for measuring the amount of the Death Receptor 5 (DR5) protein in a human biological sample of formalin-fixed tissue, comprising detecting and quantifying the level of a DR5 fragment peptide in a protein digest prepared from said human biological sample using mass spectrometry, wherein the DR5 fragment peptide has the sequence of SEQ ID NO:1; and calculating the level of modified or unmodified DR5 protein in the sample; and
wherein the amount is a relative amount or an absolute amount.
2. The method of claim 1 , further comprising the step of fractionating the protein digest prior to detecting and quantifying the level of said a DR5 fragment peptide.
3. The method of claim 1 , wherein said protein digest comprises a protease digest.
4. The method of claim 1 , wherein said formalin-fixed tissue is paraffin embedded tissue.
5. The method of claim 1 , wherein said tissue is obtained from a tumor.
6. The method of claim 1 , wherein quantifying the modified fragment peptide comprises comparing the amount of said DR5 fragment peptide in one biological sample to the amount of the same DR5 fragment peptide in a different and separate biological sample.
7. The method of claim 1 , wherein quantifying said DR5 fragment peptide comprises determining the amount of said DR5 fragment peptide in a biological sample by comparison to an added internal standard peptide of a known amount, wherein said DR5 fragment peptide in the biological sample is compared to an internal standard peptide having the same amino acid sequence, and wherein the internal standard peptide is an isotopically labeled peptide.
8. The method of claim 1 , further comprising selecting for the subject, from which the biological sample is obtained, a treatment based on the presence, absence, or amount of said DR5 fragment peptide or the amount of DR5 protein.
9. The method of claim 1 , further comprising administering to the patient from which the biological sample is 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 DR5 fragment peptide or the amount of DR5 protein.
10. The method of claim 8 , wherein the treatment or the therapeutic agent is directed to cancer cells expressing DR5 protein.