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Detection of Cystic Fibrosis Mutations by Peptide Mass Signature Genotyping

¹ Spectra Genetics, LLC, 4415 Fifth Ave., Suite 160, Pittsburgh, PA 15213.

^aAddress correspondence to this author at: University of Pittsburgh, Hillman Cancer Center, 5117 Centre Ave., Pittsburgh, PA 15213-1863. Fax 412-623-7768; e-mail malehornde{at}msx.upmc.edu.

Background: The diversity of genetic mutations and polymorphisms calls for the development of practical detection methods capable of assessing more than one patient/one nucleotide position per analysis.

Methods: We developed a new method, based on peptide mass signature genotyping (PMSG), for the detection of DNA mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Exons of the gene were amplified, cloned, and expressed in Escherichia coli as peptide fusions, in natural as well as unnatural reading frames. Peptide analytes were purified by immobilized metal affinity chromatography and analyzed by matrix-assisted, laser desorption/ionization time-of-flight mass spectrometry. Synthetic and natural DNA samples with the 25 mutations recommended for CFTR carrier screening (Grody et al. Genet Med 2001;3:149–54) were assessed using the PMSG test for the CFTR gene.

Results: Peptide analytes ranged from 6278 to 17 454 Da and varied 30-fold in expression; highly expressing peptides were observed by electron microscopy to accumulate as inclusion bodies. Peptides were reliably recovered from whole-cell lysates by a simple purification method. CFTR mutations caused detectable changes in resulting mass spectrometric profiles, which were >95% reliably detected in blinded testing of replicate synthetic heterozygous DNA samples. Mutation detection was possible with both sample pooling and multiplexing. The PMSG CFTR test was used to determine compound heterozygous mutations in DNA samples from cystic fibrosis patients, which were confirmed by direct DNA sequencing.

Conclusions: The PMSG test of the CFTR gene demonstrates unique capabilities for determining the sequence status of a DNA target by sensitively monitoring the mass of peptides, natural or unnatural, generated from that target.