High Sensitivity Measurement of Parathyroid Hormone Related Peptide Using LC-MS/MS and Associated Method

Abstract

Methods for measuring and analyzing parathyroid hormone-related peptide (PTHrP) using LC-MS/MS, including applications of the methods thereof, are disclosed and discussed. Such methods can include, along with the use of an isotope-labeled internal standard, purifying PTHrP from a biological sample, proteolytically digesting the PTHrP, and measuring specific digestion products using the using LC-MS/MS.

What is claimed is: 
     
         1 . A method of measuring parathyroid hormone-related peptide (PTHrP) in a biological sample, comprising:
 preparing the biological sample, wherein preparing further comprises:
 extracting PTHrP peptides from the biological sample; 
 mixing a PTHrP sequence-derived isotope-labeled internal standard with the PTHrP peptides; 
 proteolytically digesting the PTHrP peptides and the internal standard to produce a digestion product; and 
 chromatographically separating the digestion product; and 
   selecting and subjecting a chromatographic peak of the separated digestion product to tandem mass spectrometry to determine the concentration of a target peptide, wherein the sequence of the target peptide is selected from the group consisting of SIQDLR (SEQ ID NO: 003), AVSEHQLLHD K (SEQ ID NO: 004), FFLHHLIAEI HTAEIR (SEQ ID NO: 005), FGSDDEGR (SEQ ID NO: 006), and ATSEVSPNSK (SEQ ID NO: 007).   
     
     
         2 . The method of  claim 1 , wherein the sequence of the internal standard is AVSEHQLLHD KGKSIQDLRR RFFLHHLIAE IHTAEIRATS EVSPNSKPSP NTKNHPVRFG SDDEGRYLTQ ETNKVETYKE QPLKTP (SEQ ID NO: 001). 
     
     
         3 . The method of  claim 2 , wherein the sequence of the target peptide is SIQDLR (SEQ ID NO: 003). 
     
     
         4 . The method of  claim 1 , wherein the sequence of the target peptide is AVSEHQLLHD K (SEQ ID NO: 004). 
     
     
         5 . The method of  claim 1 , wherein the sequence of the target peptide is FFLHHLIAEI HTAEIR (SEQ ID NO: 005). 
     
     
         6 . The method of  claim 1 , wherein the sequence of the target peptide is FGSDDEGR (SEQ ID NO: 006). 
     
     
         7 . The method of  claim 1 , wherein the sequence of the target peptide is ATSEVSPNSK (SEQ ID NO: 007). 
     
     
         8 . The method of  claim 1 , wherein the internal standard is isotope-labeled in at least one amino acid with an isotope selected from the group consisting of  15 N,  13 C, and a combination thereof. 
     
     
         9 . The method of  claim 1 , wherein the internal standard is isotope-labeled in all amino acids with at least one isotope selected from the group consisting of  15 N,  13 C, and a combination thereof. 
     
     
         10 . The method of  claim 1 , wherein the internal standard is isotope-labeled in all amino acids with  15 N at all N atoms. 
     
     
         11 . The method of  claim 1 , wherein the amount of the target peptide is detectable to a sensitivity of 0.5 pmol/L or less. 
     
     
         12 . The method of  claim 1 , wherein the amount of the target peptide is detectable to a sensitivity of from 0.5 to 2.3 pmol/L. 
     
     
         13 . The method of  claim 1 , performed without a reduction reaction. 
     
     
         14 . The method of  claim 1 , performed without an alkylation reaction. 
     
     
         15 . The method of  claim 1 , wherein extracting PTHrP peptides from the biological sample further comprises an affinity extraction using an antibody selective for PTHrP. 
     
     
         16 . The method of  claim 15 , wherein the affinity extraction is antibody affinity extraction. 
     
     
         17 . The method of  claim 16 , wherein the antibody is coupled to magnetic beads. 
     
     
         18 . The method of  claim 15 , wherein the affinity extraction is aptamer affinity extraction. 
     
     
         19 . The method of  claim 1 , wherein proteolytically digesting is by trypsin digestion. 
     
     
         20 . A test sample prepared for tandem mass spectrometry, comprising:
 a trypsin-digested PTHrP peptide extracted from a biological sample; and   a PTHrP sequence-derived isotope-labeled internal standard, wherein the sequence of the PTHrP peptide and the sequence of the internal standard are the same sequence selected from the group consisting of SIQDLR (SEQ ID NO: 003), AVSEHQLLHD K (SEQ ID NO: 004), FFLHHLIAEI HTAEIR (SEQ ID NO: 005), FGSDDEGR (SEQ ID NO: 006), and ATSEVSPNSK (SEQ ID NO: 007).