Accurate characterization of the tyrosine hydroxylase forensic allele 9.3 through development of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Accurate and precise determination of the number of repeats from a short tandem repeat (STR) sequence for a human gene locus is demonstrated for the first time by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). Specifically, the polymorphic human tyrosine hydroxylase (HUMTHO1) gene, a tetranucleotide STR forensic allele, was chosen as a model system to evaluate our approach for future characterization of both STRs and variable number of tandem repeats (VNTRs) by development of an ESI-FTICR-MS approach. The coding and noncoding strands from the HUMTHO1 9.3 allele are simultaneously resolved obtaining accurate (better than 70 ppm) average mass measurements of 25,783.23 and 24,754.55 Da for the coding and noncoding strands, respectively. The mass measurements are used to calculate the number of repeats for each strand, 'n', of 9.75169 and 9.75001 for the coding and noncoding strands, respectively. It will be shown how the value of 'n' can be used to directly determine the number of pure repeats and accurately determine the exact nature of the polymorphism within the repeat (if any). The single nucleotide deletion in the coding strand (adenine) and noncoding strand (thymine) were accurately identified using this approach. Interestingly, we observed the conversion of single-stranded to double-stranded DNA while the PCR product in the ESI buffer was being infused; the issues related to this observation will be presented. Previous results by other researchers investigating the HUMTHO1 9.3 allele using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) are directly compared with our results. Our results indicate that ESI-FTICR-MS is a powerful approach to rapidly and accurately characterize tandem repeating sequences which will ultimately lead towards the understanding of a complex class of diseases and in human identity determination.