Detection of Cystic Fibrosis Mutations by Peptide Mass Signature Genotyping David E. Malehorn, * Cheryl A. Telmer, Sherri B. McEwen, Jiyan An, Ashley D. Kinsey, Adam C. Retchless, Christopher Mason, William M. Vieta, and Jonathan W. Jarvik 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 pep- tide mass signature genotyping (PMSG), for the detec- tion of DNA mutations in the cystic fibrosis transmem- brane conductance regulator (CFTR) gene. Exons of the gene were amplified, cloned, and expressed in Esche- richia coli as peptide fusions, in natural as well as unnatural reading frames. Peptide analytes were puri- fied 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 accu- mulate as inclusion bodies. Peptides were reliably re- covered 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 syn- thetic heterozygous DNA samples. Mutation detection was possible with both sample pooling and multiplex- ing. The PMSG CFTR test was used to determine com- pound heterozygous mutations in DNA samples from cystic fibrosis patients, which were confirmed by direct DNA sequencing. Conclusions: The PMSG test of the CFTR gene demon- strates 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. © 2003 American Association for Clinical Chemistry Peptide mass signature genotyping (PMSG) 1 is a genotyp- ing technology in which target DNA sequences are trans- lated to generate peptide analytes that are of suitable size for the rapid, sensitive, and specific detection of variation by matrix-assisted, laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS). This ap- proach was recently demonstrated by Garvin et al. (1), who used cell-free transcription/translation from a PCR- amplified exon of the BRCA1 gene followed by epitope- affinity capture and MALDI-TOF MS. In this report, we describe a PMSG test for mutation in the human CFTR gene. Our process differs from that of Garvin et al. (1), in several respects: (a), it uses in vivo peptide expression in Escherichia coli instead of cell-free expression; (b) the peptide analytes in our process receive their epitope tags from sequences in the plasmids from which they are expressed, instead of from 5' anchor sequences in the primers used to amplify the test se- quences; and (c) our process generates and analyzes peptides from more than one reading frame of the test sequence, a feature that allows more sensitive detection and accurate specification of the nature of changes in the DNA in many cases. Spectra Genetics, LLC, 4415 Fifth Ave., Suite 160, Pittsburgh, PA 15213. * Address 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@msx.upmc.edu. Received February 19, 2003; accepted May 23, 2003. 1 Nonstandard abbreviations: PMSG, peptide mass signature genotyping; MALDI-TOF, matrix-assisted, laser desorption/ionization time-of-flight; MS, mass spectrometry; CFTR, cystic fibrosis transmembrane conductance regula- tor; UE, universal epitope; IPTG, isopropylthio--galactoside; SDS, sodium dodecyl sulfate; ACMG, American College of Medical Genetics; EA, epon- araldite; IMAC, immobilized metal affinity chromatography; and PAGE, polyacrylamide gel electrophoresis. Clinical Chemistry 49:8 1318 –1330 (2003) Molecular Diagnostics and Genetics 1318 Downloaded from https://academic.oup.com/clinchem/article/49/8/1318/5641800 by guest on 26 December 2022