Methods of detecting and treating pulmonary hypertension

Abstract

The present disclosure describes a method of quantifying amounts of phosphopeptides using isotopically-enriched peptides as internal standards. A kit comprising at least one isotopically-enriched phosphorylated peptide can be used to quantify changes in amounts of phosphopeptides using parallel reaction monitoring mass spectrometry techniques. The invention can be used to indicate the pathologic mechanism, severity of the disease, and treatment response of a subject. The invention can also be used to identify subjects who require more aggressive therapeutic interventions or alternative treatments.

What is claimed is: 
     
         1 . A method comprising:
 a) adding to a sample of a protein extracted from a buffy coat an amount of a quantification standard to provide a test sample; and   b) assaying the test sample to quantify in the test sample a wild type phosphopeptide of the protein extracted from the buffy coat, wherein the quantifying is based on the amount of the quantification standard, wherein the quantification standard is a form of the phosphopeptide that is isotopically enriched to a non-natural abundance of an isotope.   
     
     
         2 . The method of  claim 1 , further comprising:
 i) obtaining a tissue sample from a human subject, wherein the tissue sample contains the buffy coat; and   ii) isolating the protein from the tissue sample to provide the sample of the protein.   
     
     
         3 . The method of  claim 2 , further comprising digesting the sample of protein prior to adding the amount of the quantification standard. 
     
     
         4 . The method of  claim 2 , wherein the human subject has pulmonary hypertension. 
     
     
         5 . The method of  claim 2 , wherein the human subject has pulmonary arterial hypertension. 
     
     
         6 . The method of  claim 4 , wherein the pulmonary hypertension is idiopathic pulmonary hypertension. 
     
     
         7 . The method of  claim 4 , wherein the pulmonary hypertension is pulmonary hypertension associated with systemic sclerosis. 
     
     
         8 . The method of  claim 1 , wherein the quantification standard is an isotopically-enriched peptide of TABLE 2 or TABLE 3. 
     
     
         9 . The method of  claim 1 , wherein the quantification standard is an isotopically-enriched peptide of TABLE 6 or TABLE 7. 
     
     
         10 . The method of  claim 1 , wherein the quantifying is performed by mass spectrometry. 
     
     
         11 . The method of  claim 1 , wherein the quantifying is performed by parallel reaction monitoring mass spectrometry. 
     
     
         12 . The method of  claim 1 , wherein the quantifying is performed by contacting the test sample with a phospho-specific antibody. 
     
     
         13 . The method of  claim 1 , wherein the quantifying comprises detecting a ratio of the wild type phosphopeptide amount to an amount of a non-phosphorylated form of the wild type peptide. 
     
     
         14 . The method of  claim 13 , wherein the ratio of the amount of the wild type phosphopeptide to the amount of the non-phosphorylated form of the wild type peptide is greater than a corresponding ratio obtained from a control sample obtained from a test subject that has pulmonary arterial hypertension. 
     
     
         15 . The method of  claim 13 , wherein the ratio of the amount of the wild type phosphopeptide to the amount of the non-phosphorylated form of the wild type peptide is less than a corresponding ratio obtained from a control sample obtained from a test subject that has pulmonary arterial hypertension. 
     
     
         16 . The method of  claim 13 , wherein the ratio of the amount of the wild type phosphopeptide to the amount of the non-phosphorylated form of the wild type peptide is greater than a corresponding ratio obtained from a test subject that does not have pulmonary arterial hypertension. 
     
     
         17 . The method of  claim 13 , wherein the ratio of the amount of the wild type phosphopeptide to the amount of the non-phosphorylated form of the wild type peptide is less than a corresponding ratio obtained from a test subject that does not have pulmonary arterial hypertension. 
     
     
         18 . A chemical aliquot comprising a sample of a phosphopeptide, wherein at least 1% of the sample of the phosphopeptide consists of molecules that have an amino acid residue that has at least two  13 C nuclei, wherein the sample contains at least 1 pg of the phosphopeptide, wherein the phosphopeptide has at least 90% sequence identity to any compound of TABLE 2 or TABLE 3. 
     
     
         19 . The chemical aliquot of  claim 18 , wherein at least 10% of the sample of the phosphopeptide consists of molecules that have the amino acid residue that has at least two  13 C nuclei. 
     
     
         20 . The chemical aliquot of  claim 18 , wherein at least 50% of the sample of the phosphopeptide consists of molecules that have the amino acid residue that has at least two  13 C nuclei. 
     
     
         21 . The chemical aliquot of  claim 18 , wherein the amino acid residue that has at least two  13 C nuclei has at least three  13 C nuclei. 
     
     
         22 . The chemical aliquot of  claim 18 , wherein the amino acid residue that has at least two  13 C nuclei has six  13 C nuclei. 
     
     
         23 . The chemical aliquot of  claim 18 , wherein the amino acid residue that has at least two  13 C nuclei has at least two  15 N nuclei. 
     
     
         24 . The chemical aliquot of  claim 18 , wherein the amino acid residue that has at least two  13 C nuclei is lysine. 
     
     
         25 . The chemical aliquot of  claim 18 , wherein the amino acid residue that has at least two  13 C nuclei is arginine. 
     
     
         26 . A kit comprising:
 a) a chemical aliquot comprising a sample of a phosphopeptide, wherein at least 1% of the sample of the phosphopeptide consists of molecules that have an amino acid residue that has at least two  13 C nuclei, wherein the sample contains at least 1 pg of the phosphopeptide, wherein the phosphopeptide has at least 90% sequence identity to any compound of any of TABLE 2 or TABLE 3; and   b) an additional aliquot comprising a sample of the phosphopeptide that has nuclei in a natural abundance of isotopes.