Mass spectrometric detection of polypeptides

Abstract

A process for ascertain sequence information about a nucleic acid molecule by determining the identity of a polypeptide using mass spectroscopy is provided. Depending on the polypeptide to be identified, a process as disclosed is used, for example, to diagnose a genetic disease or chromosomal abnormality, a predisposition to a disease or condition, or infection by a pathogenic organism; or for determining identity or heredity. Kits for performing the disclosed processes also are provided.

What is claimed is:  
     
         1 . A kit for determining the identity of a target nucleic acid by mass spectrometry, comprising 
 a) a forward and a reverse primer that hybridizes to a nucleic acid encoding the target polypeptide for amplifying the nucleic acid;    b) reagents for in vitro transcription and translation of the amplified nucleic acid to obtain the encoded polypeptide; and    c) a reagent for isolating the polypeptide;    d) a reagent for mass spectrometric analysis of the polypeptide; and    d) optionally, instructions for use in determining the identity of a target polypeptide by mass spectrometry to thereby obtain sequence information about the nucleic acid.    
     
     
         2 . A kit of  claim 1 , wherein the polypeptide is encoded by an allelic variant of a polymorphic region of a gene of a subject.  
     
     
         3 . A kit of  claim 2 , for determining whether a subject has or is at risk of developing a disease or condition associated with a specific allelic variant of a polymorphic region of the gene.  
     
     
         4 . A kit of  claim 3 , wherein the disease or condition is associated with an aberrant number of trinucleotide repeats.  
     
     
         5 . A kit of  claim 4 , wherein the disease or condition is selected from the group consisting of Huntington's disease, prostate cancer, Fragile X syndrome type A, myotonic dystrophy type I, Kennedy disease, Machado-Joseph disease, Dentatorubral and pallidolyusian atrophy and spino bulbar muscular atrophy.  
     
     
         6 . A kit of  claim 3 , wherein the gene is selected from the group consisting of BRCA1, BRCA2, APC, dystrophin gene, β-globin, Factor IX, Factor VIIc, ornithine-d-amino-transferase, hypoxanthine guanine phosphoribosyl transferase, CFTR, and a proto-oncogene.  
     
     
         7 . A kit of  claim 1 , wherein the forward primer and/or the reverse primer comprises an RNA polymerase promoter.  
     
     
         8 . A kit of  claim 7 , wherein the RNA polymerase promoter is selected from the group consisting of SP6 promoter, T3 promoter, and T7 promoter.  
     
     
         9 . A kit of  claim 7 , wherein the forward and/or the reverse primer further comprises a nucleotide sequence, or complement thereof, encoding a second polypeptide.  
     
     
         10 . A kit of  claim 9 , wherein the second polypeptide is a Tag polypeptide.  
     
     
         11 . A kit of  claim 10  , wherein the Tag polypeptide is selected from the group consisting of a myc-epitope tag, a  Haemophilys influenza  hemagglutinin protein tag and a HIS-6.  
     
     
         12 . A kit of  claim 10 , wherein the Tag polypeptide is HIS-6 and the reagent for isolating the polypeptide is nickel ion.  
     
     
         13 . A kit of  claim 1 , further comprising a reference nucleic acid or polypeptide.  
     
     
         14 . A kit of  claim 1 , wherein the reagent for mass spectrometric analysis comprises a reagent system for volatilizing and ionizing the polypeptide prior to mass spectrometric analysis.  
     
     
         15 . A kit of  claim 14 , wherein the reagent system comprises an organic solvent.  
     
     
         16 . The kit of  claim 14 , wherein the reagent system comprises an inorganic solvent.  
     
     
         17 . The kit of  claim 16 , wherein the inorganic reagent system comprises an ammonium salt.  
     
     
         18 . The kit of  claim 1 , wherein either the forward primer or the reverse primer comprises a sequence of nucle.otides that, following amplification, encode a regulatory element operably linked to the nucleic acid encoding the target polypeptide.  
     
     
         19 . The kit of  claim 18 , wherein the regulatory element is selected from the group consisting of an RNA polymerase promoter, a ribosome binding site, a START codon, and a transcription start signal.