Mutation scanning by ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS).
Partially denaturing high-performance liquid chromatography has emerged as the most sensitive physical mutation scanning method. However, there are a few reports of mutations missed or only detectable at unique temperatures. The combined use of ion-pair reversed-phase high-performance liquid chromatography under completely denaturing conditions and electrospray ionization quadrupole ion trap mass spectrometry (ICEMS) obviates the need for selecting appropriate temperatures for resolving heteroduplices and allows the discrimination of different alleles even when they co-elute due to distinct mass differences between nucleobases. This was demonstrated for the detection of four mutations (259G>A, 286A>G, 300T>G, and 331+1G>A) in exon 5 and intron 5 of BRCA1, respectively. Current mass resolution of quadrupole ion trap mass spectrometers limits the identification of single A>T or T>A transversions with a mass difference of 9 Da to fragments <80 base pairs (bp). The presence of all other mutations can be detected in fragments up to approximately 105 bp. The approach may prove particularly useful in the mutational scanning of AT- or GC-rich sequences that are recalcitrant to most other methods.
Oberacher
Herbert
H
Instrumental Analysis and Bioanalysis, Saarland University, Saarbrücken, Germany.
Huber
Christian G
CG
Oefner
Peter J
PJ
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D019398:Genes, BRCA1; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D020641:Polymorphism, Single Nucleotide; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*