Secreted Protein Acidic and Rich in Cysteine (SPARC) Protein SRM/MRM Assay

Abstract

The current disclosure provides for specific peptides, and derived ionization characteristics of the peptides, from the Secreted Protein Acidic and Rich in Cysteine (SPARC) protein that are particularly advantageous for quantifying the SPARC 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 wherein said 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 said biological sample using the Liquid Tissue™ reagents and protocol and the SPARC 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 SPARC peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

1 - 20 . (canceled) 
     
     
         21 . A method for measuring the amount of the Secreted Protein Acidic and Rich in Cysteine (SPARC) protein in a human biological sample of formalin-fixed tissue, comprising detecting and quantifying using mass spectrometry the amount of a SPARC fragment peptide in a protein digest prepared from said human biological sample, wherein the SPARC fragment peptide has the sequence of SEQ ID NO:5; and calculating the level of modified or unmodified DR5 protein in the sample; wherein the amount is a relative amount or an absolute amount, wherein detecting and quantifying the amount of said SPARC fragment peptide in the protein digest indicates the presence of SPARC protein and an association with cancer in the subject. 
     
     
         22 . The method of  claim 21 , further comprising correlating the results of said detecting and quantifying the amount of said SPARC fragment peptide, or the level of said SPARC protein to the diagnostic stage/grade/status of the cancer. 
     
     
         23 . The method of  claim 22 , wherein correlating the results of said detecting and quantifying the amount of said SPARC fragment peptide, or the level of said SPARC 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. 
     
     
         24 . The method of  claim 21 , further comprising selecting for the subject from which said biological sample was obtained a treatment based on the presence, absence, or amount of said SPARC fragment peptide or the level of SPARC protein. 
     
     
         25 . The method of  claim 21 , further comprising administering to the patient 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 SPARC fragment peptide or the level of SPARC protein. 
     
     
         26 . The method of  claim 25 , wherein said therapeutic agent is directed to cancer cells expressing SPARC protein. 
     
     
         27 - 31 . (canceled) 
     
     
         32 . The method of  claim 24 , wherein said treatment comprises a therapeutic agent directed to cancer cells expressing SPARC protein