Methods and materials for monitoring myeloma using quantitative mass spectrometry

Abstract

The subject invention concerns methods and materials for diagnosing, monitoring the progress, and/or providing a prognosis for multiple myeloma and other conditions associated with antibody production in a person or animal. The methods o f the invention utilize mass spectrometry for quantitative monitoring and detection of antibody produced by the plasma cells. The methods of the invention can be utilized for diagnosis, monitoring, and/or prognosis of multiple myeloma, monoclonal gammopathy, and other immunological or hematological conditions and disorders. In addition to detecting and quantifying antibody in a sample, other biological markers, such as serum albumin and/or beta-2-microglobulin, can also be detected and quantified using the present invention, and in combination with detection and quantification of antibody. Thus, in one embodiment, both antibody and serum albumin and/or beta-2-microglobulin are detected and quantified using mass spectrometry and a diagnosis or prognosis made based on the results and levels detected.

1 . An isolated peptide comprising an amino acid sequence shown in any of SEQ ID NO:1 to SEQ ID NO:30, or a fragment thereof. 
     
     
         2 . The isolated peptide according to  claim 1 , wherein the peptide consists of an amino acid sequence shown in any of SEQ ID NO:1 to SEQ ID NO:30, or a fragment thereof. 
     
     
         3 . The isolated peptide according to  claim 1 , wherein the peptide comprises a stable heavy isotope. 
     
     
         4 . A method for diagnosing, monitoring the progress of, and/or providing a prognosis of a disorder or condition associated with antibody production in a person or animal, said method comprising treating a biological sample from the person or animal to isolate a target protein; fragmenting the target protein to create peptide fragments thereof; subjecting the peptide fragments of the target protein to quantitative mass spectrometry to identify and quantitate the amount of target protein in the biological sample; and correlating the identity and quantity of the target protein in the biological sample to a disorder or condition in the person or animal, whereby the disorder or condition can be diagnosed, monitored for progress, and/or a prognosis provided. 
     
     
         5 . The method according to  claim 4 , wherein the target protein is an antibody. 
     
     
         6 . The method according to  claim 4 , wherein the target protein is a serum albumin and/or a beta-2-microglobulin. 
     
     
         7 . The method according to  claim 5 , wherein the antibody comprises human heavy chain of IgG, IgA, IgM, IgD, or IgE. 
     
     
         8 . The method according to  claim 5 , wherein the antibody comprises human kappa light chain or human lambda light chain. 
     
     
         9 . The method according to  claim 4 , wherein the disorder or condition is multiple myeloma or monoclonal gammopathy of undetermined significance (MGUS). 
     
     
         10 . The method according to  claim 4 , wherein the target protein is identified and quantitated by spiking in a known amount of a specific labeled peptide corresponding to a fragment of a target protein during mass spectrometry, wherein the specific labeled peptide comprises a heavy isotope label or has an amino acid substitution to create a mass difference between the fragment of the target protein and the specific labeled peptide. 
     
     
         11 . The method according to  claim 10 , wherein the heavy isotope label is  2 H,  13 C, or  15 N. 
     
     
         12 . The method according to  claim 10 , wherein the specific labeled peptide comprises an amino acid sequence of any of SEQ ID NO:1 to SEQ ID NO:30. 
     
     
         13 . The method according to  claim 10 , wherein the specific labeled peptide corresponds to a fragment of a human antibody heavy chain of IgG, IgA, IgM, IgD, or IgE. 
     
     
         14 . The method according to  claim 10 , wherein the specific labeled peptide corresponds to a fragment of a human antibody heavy chain of IgG and comprises the amino acid sequence ALPAPIEK (SEQ ID NO:4). 
     
     
         15 . The method according to  claim 10 , wherein the specific labeled peptide corresponds to a fragment of a human antibody kappa light chain or lambda light chain. 
     
     
         16 . The method according to  claim 10 , wherein the specific labeled peptide corresponds to a fragment of a human antibody kappa light chain and comprises the amino acid sequence DSTYSLSSTLTLSK (SEQ ID NO:28). 
     
     
         17 . The method according to  claim 10 , wherein the specific labeled peptide corresponds to a fragment of a human serum albumin or human beta-2-microglobulin. 
     
     
         18 . The method according to  claim 4 , wherein the target protein is denatured prior to fragmentation. 
     
     
         19 . The method according to  claim 18 , wherein the target protein is denatured by treatment with urea, disulfide reduction, and/or cysteine alkylation. 
     
     
         20 . The method according to  claim 4 , wherein the target protein is fragmented by proteolytic enzyme digestion. 
     
     
         21 . The method according to  claim 20 , wherein the proteolytic enzyme is trypsin. 
     
     
         22 . The method according to  claim 4 , wherein said treating step comprises one or more of size exclusion chromatography, gel electrophoresis, and/or affinity chromatography to isolate the target protein. 
     
     
         23 . The method according to  claim 4 , wherein the mass spectrometry comprises liquid chromatography coupled to multiple reaction monitoring (LC-MRM). 
     
     
         24 . The method according to  claim 23 , wherein the mass spectrometry is conducted on a triple quadrupole mass spectrometer. 
     
     
         25 . The method according to  claim 10 , wherein the amino acid substitution comprises substitution of alanine for glycine. 
     
     
         26 . The method according to  claim 4 , wherein the method is used to monitor for progression of monoclonal gammopathy of undetermined significance in the person or animal to multiple myeloma; or wherein the method is used to monitor the efficacy of a treatment regimen on a person or animal with multiple myeloma or MGUS. 
     
     
         27 . (canceled) 
     
     
         28 . The isolated peptide according to  claim 2 , wherein the peptide comprises a stable heavy isotope.