Secreted protein acidic and rich in cysteine (SPARC) protein SRM assay
The current disclosure provides for specific peptides from the Secreted Protein Acidic and Rich in Cysteine (SPARC) protein and the derived ionization characteristics of those peptides that are advantageous for quantifying the SPARC directly in formalin fixed biological samples by the method of Selected Reaction Monitoring (SRM) mass spectrometry. Such fixed biological samples include: formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and formalin fixed and paraffin embedded tissue culture cells. SPARC protein is quantitated in biological samples by the method of SRM/MRM mass spectrometry by quantitating one or more of the peptides described herein. The peptides can be quantitated if they reside in a modified or an unmodified form. Examples of potentially modified forms of an SPARC peptides include those bearing 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 Secreted Protein Acidic and Rich in Cysteine (SPARC) protein in a human biological sample of formalin-fixed tissue, comprising detecting and quantifying the amount of a SPARC fragment peptide in a protein digest prepared from said biological sample using mass spectrometry;
and using said amount of said SPARC fragment peptide to calculate the level of SPARC protein in said sample; wherein the SPARC fragment peptide consists of the peptide of SEQ ID NO:5 and
wherein said level is a relative level or an absolute level.
2. The method of claim 1 , further comprising the step of fractionating said protein digest prior to detecting and quantifying the amount of said SPARC fragment peptide.
3. The method of claim 1 , wherein said 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 the SPARC fragment peptide comprises comparing an amount of said SPARC fragment peptide in one biological sample to the amount of the same SPARC fragment peptide in a different and separate biological sample.
7. The method of claim 1 , wherein quantifying said SPARC fragment peptide comprises determining the amount of said SPARC fragment peptide in a biological sample by comparison to an added internal standard peptide of known amount,
wherein said SPARC 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.