Mass spectrometric immunoassay analysis of specific proteins and variants present in various biological fluids

Abstract

Presented herein is the construction of pipettor tips (termed MSIA-Tips) containing porous solid supports that are constructed, covalently derivatized with affinity ligand, and used to extract specific proteins and their variants from various biological fluids by repeatedly flowing the fluids through the MSIA-Tips. A second protein species (a mass-shifted variant of the targeted protein doped into the samples at a constant concentration) is co-extracted with the endogenous protein and variants and is used as a quantitative internal reference standard (IRS). Nonspecifically bound compounds are rinsed from the MSIA-Tip using a series of buffer and water rinses, after which the wild type protein, protein variants and the IRS are eluted from the MSIA-Tips directly onto a target in preparation for analysis such as MALDI-TOF. Mass spectrometry of the eluted sample then follows with the retained proteins identified via accurate molecular mass determination. Protein and variant levels are determined via a quantitative method in which the protein/variant signals are normalized to the signal of the IRS and the values compared to a working curve constructed from samples containing known concentrations of the protein or variants.

What is claimed is:  
     
         1 . A kit for the isolation and subsequent qualitative or quantitative characterization of target biomolecules present in biological fluid comprising:at least one MSIA-Tip having an affinity reagent present within the tip, at least one internal reference standard of predetermined concentration, and at least one mass spectrometer target.  
     
     
         2 . The kit according to  claim 1  wherein the affinity reagent further comprises an affinity ligand, said affinity ligand further comprises anti-human β-2-microglobulin antibody.  
     
     
         3 . The kit according to  claim 1  wherein the internal reference standard is an internal reference standard that shares sequence homology with the target biomolecule.  
     
     
         4 . The kit according to  claim 3  wherein the internal reference standard that shares sequence homology with the target biomolecule is selected from the group comprising enzymatic/chemically-modified versions of the target biomolecule, truncated/extended recombinant forms of the target biomolecules, the target biomolecule recombinantly expressed in isotopically-enriched media, and the target biomolecule from a different biological species.  
     
     
         5 . The kit according to  claim 3  wherein the internal reference standard that shares sequence homology with the target biomolecule is equine β-2-microglobulin.  
     
     
         6 . The kit according to  claim 2  wherein the internal reference standard is an internal reference standard that shares sequence homology with the target biomolecule.  
     
     
         7 . The kit according to  claim 6  wherein the internal reference standard that shares sequence homology with the target biomolecule is selected from the group comprising enzymatic/chemically-modified versions of the target biomolecule, truncated/extended recombinant forms of the target biomolecules, the target biomolecule recombinantly expressed in isotopically-enriched media, and the target biomolecule from a different biological species:  
     
     
         8 . The kit according to  claim 6  wherein the internal reference standard that shares sequence homology with the target biomolecule is equine β-2-microglobulin.  
     
     
         9 . A method for the isolation and subsequent qualitative characterization of target biomolecules present in biological fluid comprising the steps of: 
 a. providing a MSIA-Tip having an affinity reagent present,    b. separating and concentration the target biomolecule directly from the biological fluid by flowing a volume of the biological fluid through the MSIA-Tip, thereby binding the target biomolecules to the affinity reagent,    c. cluting the target biomolecules onto a mass spectrometer target,    d. performing mass spectrometric analysis on the target biomolecules in order to qualitatively determine the presence or absence of the target biomolecule in the biological fluid.    
     
     
         10 . The method according to  claim 9  wherein the affinity reagent further comprises an affinity ligand, said affinity ligand further comprises anti-human β-2-microglobulin antibody.  
     
     
         11 . The method according to  claim 9  wherein the qualitative determination further determines the presence of mass shifted variants of the target biomolecule.  
     
     
         12 . The method according to  claim 10  wherein the qualitative determination further determines the presence of mass shifted variants of the target biomolecule.  
     
     
         13 . A method for the isolation and subsequent quantitative characterization of target biomolecules present in biological fluid comprising the steps of: 
 a. adding a known amount of internal reference standard of predetermined concentration to a sample of the biological fluid,    b. providing a MSIA-Tip having an affinity reagent present,    c. flowing a volume of the biological fluid through the pipettor tip, thereby binding the target biomolecules to the affinity reagent,    d. eluting the target biomolecules to a mass spectrometer target,    e. performing mass spectrometric analysis on the target biomolecules in order to quantitatively determine the concentration of the target biomolecule in the biological fluid.    
     
     
         14 . The method according to  claim 13  wherein the affinity reagent further comprises an affinity ligand, said affinity ligand further comprises anti-human β-2-microglobulin antibody.  
     
     
         15 . The method according to  claim 13  wherein the internal reference standard is an internal reference standard that shares sequence homology with the target biomolecule.  
     
     
         16 . The method according to  claim 15  wherein the internal reference standard that shares sequence homology with the target biomolecule is selected from the group comprising enzymatic/chemically-modified versions of the target biomolecule, truncated/extended recombinant forms of the target biomolecules, the target biomolecule recombinantly expressed in isotopically-enriched media, and the target biomolecule from a different biological species.  
     
     
         17 . The method according to  claim 15  wherein the internal reference standard that shares sequence homology with the target biomolecule is equine β-2-microglobulin.  
     
     
         18 . The method according to  claim 14  wherein the internal reference standard is an internal reference standard that shares sequence homology with the target biomolecule.  
     
     
         19 . The method according to  claim 18  wherein the internal reference standard that shares sequence homology with the target biomolecule is selected from the group comprising enzymatic/chemically-modified versions of the target biomolecule, truncated/extended recombinant forms of the target biomolecules, the target biomolecule recombinantly expressed in isotopically-enriched media, and the target biomolecule from a different biological species.  
     
     
         20 . The method according to  claim 18  wherein the internal reference standard that shares sequence homology with the target biomolecule is equine β-2-microglobulin.  
     
     
         21 . The method according to  claim 13  wherein the quantitative determination further determines the concentration of mass shifted variants of the target biomolecule.  
     
     
         22 . The method according to  claim 14  wherein the quantitative determination further determines the concentration of mass shifted variants of the target biomolecule.  
     
     
         23 . The method according to  claim 15  wherein the quantitative determination further determines the concentration of mass shifted variants of the target biomolecule.  
     
     
         24 . The method according to  claim 16  wherein the quantitative determination further determines the concentration of mass shifted variants of the target biomolecule.  
     
     
         25 . The method according to  claim 17  wherein the quantitative determination further determines the concentration of mass shifted variants of the target biomolecule.  
     
     
         26 . The method according to  claim 18  wherein the quantitative determination further determines the concentration of mass shifted variants of the target biomolecule.  
     
     
         27 . The method according to  claim 19  wherein the quantitative determination further determines the concentration of mass shifted variants of the target biomolecule.  
     
     
         28 . The method according to  claim 20  wherein the quantitative determination further determines the concentration of mass shifted variants of the target biomolecule.