MALDI-TOF Mass Spectrometric Detection of Multiplex Single Base Extended Primers. A Study of 17 Y-Chromosome Single-Nucleotide Polymorphisms

Abstract

One of the most promising techniques for typing of multiple single-nucleotide polymorphism (SNP) is detection of single base extension primers (SBE) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We present a new MALDI-TOF MS protocol for typing of multiple SNPs in a single reaction. Biotin-labeled ddNTPs were used in the SBE reaction and solid phase-bound monomeric avidin was used as capturing/purification scheme allowing the exclusive release of the SBE products under gentle conditions using 5% triethylamine. We dubbed this method monomeric avidin triethylamine purification. The biotin-labeled ddNTPs contained linkers with different masses ensuring a clear separation of the alleles even for SBE primers with a mass of 10 300 Da. Furthermore, only 25-350 fmol of SBE primers were necessary in order to obtain reproducible MALDI-TOF spectra. Similar signal intensities were obtained in the 5500-10 300 m/z mass range by increasing the concentration of the longer SBE primers in the reaction. To validate the technique, 17 Y-chromosome SNPs were analyzed in 200 males. The precision and accuracy of the mass determination were analyzed by parametric statistic, and the potential use of MALDI-TOF MS for SNP typing is discussed.

Standardized Peptidome Profiling of Human Urine by Magnetic Bead Separation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

“…One reason for the improved precision of MB-MALDI-TOF MS might be the application of 4-fold spotting of the MALDI target for each urine sample. The determination of the mass accuracy (M r range, 1000 -10 000) produced absolute differences in M r of 0.35-3.5, which are comparable to our data for serum and in accordance with the known mass accuracy of MALDI-TOF MS (29,31 ).…”

Standardized Approach to Proteome Profiling of Human Serum Based on Magnetic Bead Separation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

“…The mean mass shift of 0.035% led to absolute mass differences between 0.35 and 3.5 Da in the mass range 1000 -10 000 Da. These absolute mass differences are in accordance with the known mass accuracy of MALDI-TOF MS (24 ). We solved the problem of mass shift by using the most prominent peaks to recalibrate the spectra with the ClinProTools bioinformatics software (19 ).…”

Simple, Efficient, and Cost-Effective Multiplex Genotyping with Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of Hemoglobin Beta Gene Mutations

“…19,[22][23][24]32,34,35 Solid phase capturable ddNTPs in SBE have been developed using biotinylated ddNTPs to generate 3Ј-biotinylated extension DNA products, which then are purified by streptavidin-coated magnetic beads before analysis by MS. 27 This solid phase capturable-SBE method has been applied for simultaneous genotyping of 17 Y-chromosome SNPs, 36 detection of 30 point mutations in p53 in a single tube, 37 and for concurrent analysis of 40 SNPs of CYP2C9 and 50 SNPs of CYP2A13 genes. 38 However, the cost-effectiveness of this approach has not been evaluated.…”