|Abstract:|| Specific peptides, and derived ionization characteristics of those peptides, from the Bcl-2-like protein 11 (BIM) are provided that are particularly advantageous for quantifying the BIM 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). Such biological samples are chemically preserved and fixed where 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.TM. reagents and protocol, and the BIM protein is quantitated in the Liquid Tissue.TM. 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 a BIM peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.|
|Inventor(s):|| Krizman; David (Gaithersburg, MD), Hembrough; Todd (Gaithersburg, MD), Thyparambil; Sheeno (Frederick, MD), Liao; Wei-Li (Herndon, VA) |
|Patent Claims:||1. A method for measuring the level of the human Bcl-2-like protein 11 (BIM) in a human biological sample of formalin-fixed tissue, comprising detecting and
quantifying the amount of a BIM fragment peptide in a protein digest prepared from said biological sample using mass spectrometry; and calculating the amount of BIM protein in said sample; wherein the BIM fragment peptide consists of the peptide of SEQ
ID NO:2 and wherein said level is a relative level or an absolute level.
2. The method of claim 1, further comprising the step of fractionating the protein digest prior to detecting and/or quantifying the level of said BIM fragment peptide.
3. The method of claim 1, wherein the protein digest comprises a protease digest.
4. The method of claim 1, wherein the tissue is paraffin embedded tissue.
5. The method of claim 1, wherein the tissue is obtained from a tumor.
6. The method of claim 1, wherein quantifying said fragment peptide comprises comparing the level of said BIM fragment peptide in one biological sample to the level of the same BIM fragment peptide in a different and separate biological sample.
7. The method of claim 6, wherein quantifying said BIM fragment peptide comprises determining the level of said BIM fragment peptide in a biological sample by comparison to an added internal standard peptide of a known level, wherein said BIM
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 administering to the patient or subject, 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 level of said BIM fragment peptide or the level of BIM protein, and wherein said therapeutic agent is a kinase receptor inhibitor.
9. The method of claim 8, wherein the treatment or the therapeutic agent is directed to cancer cells expressing BIM protein.
10. The method of claim 8, wherein said therapeutic agent is an antibody that binds a kinase receptor.
11. The method of claim 10 wherein said kinase receptor inhibitor is trastuzumab, cetuximab, or panitumumab.
12. The method of claim 8 wherein said kinase receptor inhibitor is selected from the group consisting of Lapatinib, Erlotinib, Gefitinib, Vandetanib, Pelitinib, Canertinib, Foretinib, Crizotinib, Afatinib, Cabozantinib, Axitinib, Vatalanib,
BMS-536924, OSI-906, Saracatinib, and Ponatinib.