SRM/MRM assay for the ephrin type-A receptor 2 protein
Specific peptides, and derived ionization characteristics of the peptides, from the Ephrin Type-A Receptor 2 (EPHA2) protein are provided that are particularly advantageous for quantifying the EPHA2 protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed and are 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 said biological sample using the Liquid Tissue™ reagents and protocol and the EPHA2 protein is 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 EPHA2 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 Ephrin Type-A Receptor 2 (EPHA2) protein in a human biological sample of formalin-fixed tumor tissue, comprising detecting and quantifying the amount of an unmodified EPHA2 fragment peptide in a protease digest prepared from said biological sample using mass spectrometry; and calculating the level of EPHA2 protein in said sample; wherein the EPHA2 fragment peptide consists of SEQ ID NO:1, and wherein said amount is an absolute amount.
2. The method of claim 1 , further comprising the step of fractionating said protease digest prior to detecting and/or quantifying the amount of said EPHA2 fragment peptide.
3. The method of claim 2 , wherein said fractionating step is selected from the group consisting of gel electrophoresis, liquid chromatography, capillary electrophoresis, nano-reversed phase liquid chromatography, high performance liquid chromatography, or reverse phase high performance liquid chromatography.
4. The method of claim 1 , wherein said protease digest comprises a trypsin 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 5 , wherein the mode of mass spectrometry used is Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), and/or multiple Selected Reaction Monitoring (mSRM), or any combination thereof.
7. The method of claim 1 , wherein the tissue is paraffin embedded tissue.
8. The method of claim 1 , wherein quantifying said EPHA2 fragment peptide comprises determining the amount of said EPHA2 fragment peptide in a biological sample by comparison to an added internal standard peptide of known amount, wherein said EPHA2 fragment peptide in the biological sample is compared to an internal isotopically labeled standard peptide having the same amino acid sequence.
9. The method of claim 8 , wherein the isotopically labeled internal standard peptide comprises one or more heavy stable isotopes selected from 18 O, 17 O, 34 S, 15 N, 13 C, 2 H or combinations thereof.
10. The method of claim 1 , wherein said tumor is a prostate tumor.
11. The method of claim 1 , wherein said tumor is a colon tumor.