Insulin receptor substrate 1 (IRS1) protein SRM/MRM assay
The current disclosure provides for specific peptides from the Insulin Receptor Substrate 1 (IRS1) protein and the derived ionization characteristics of those peptides that are advantageous for quantifying IRS1 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. IRS1 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 IRS1 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 Insulin Receptor Substrate 1 (IRS 1) protein in a human biological sample of formalin fixed tissue, comprising detecting and quantifying the amount of an IRS 1 fragment peptide in a protein digest prepared from said biological sample using mass spectrometry; and calculating the level of IRS 1 protein in said sample; wherein said protein digest comprises a protease digest, wherein the tissue is obtained from a tumor, wherein said IRS 1 fragment peptide has the sequence set forth in SEQ ID NO:76, wherein quantifying said IRS 1 fragment peptide comprises determining the amount of said IRS 1 fragment peptide in a biological sample by comparison to an added internal standard peptide of known amount, wherein said IRS 1 fragment peptide in the biological sample is compared to an internal standard peptide having the same amino acid sequence, wherein the internal standard peptide is an isotopically labeled peptide, and wherein said level of IRS1 protein in said biological sample is 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 IRS1 fragment peptide.
3. The method of claim 1 , wherein the tissue is paraffin embedded tissue.