MS for Identification of Single Nucleotide Polymorphisms and MS/MS for Discrimination of Isomeric PCR Products

Abstract

ESI (electrospray ionization) MS and tandem mass spectrometry (MS/MS) were used for the analysis of single nucleotide polymorphisms (SNPs) and more complex genetic variations. Double-stranded (ds) PCR products were studied. PCR products of the proline [5'-x(G17)-x(C38)x-3'] and arginine variants [(5'-x(Gl7)-x(G38)x-3'] of the p53 gene are distinguished by an SNP (cytosine or guanine) and were discriminated using both quadrupole and quadrupole ion trap MS analysis. A 69 bp arginine mutant PCR product [5'-x(C17)-x(G38)x-3'] with a negating switch has the same mass as the proline variant but was readily distinguishable on ion trap MS/MS analysis; fragments containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products were restriction-enzyme-digested, to create 43 bp fragments. ESI quadrupole ion trap MS/MS analysis of the 43 bp product-ion spectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS are powerful and complementary techniques for analysis of DNA. MS can readily distinguish SNPs but MS/MS is required to differentiate isomeric PCR products (same nucleotide composition but different sequence).

Multi-charged Oligonucleotide Ion Formation in Sonic Spray Ionization

“…At rates higher than 80 µL/min, the situation was reversed. The ion state of (M-6H) 6-even (M-5H) 5-dominated the oligonucleotide ions.…”

“…9). A large amount of protons dissociated from the acetic acid greatly depressed the highly charged ions, (M-7H) 7-and (M-6H) 6-. Thus, the chemical effect plays an important role for oligonucleotide ion formation.…”

“…Although MS only offers short read lengths for DNA sequencing currently, compared with other conventional DNA sequencing methods, this method can deal with SNPs analysis without any large problems in its mass range. Among the published works, [2][3][4][5][6][7] most were carried out with a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. An alternative way is to spray a sample solution and thus make DNA ionized by electrospray ionization (ESI) 7 or sonic spray ionization (SSI).…”

Factors determining the performance of triple quadrupole, quadrupole ion trap and sector field mass spectrometers in electrospray ionization tandem mass spectrometry of oligonucleotides. 1. Comparison of performance characteristics

“…For preparation of all solutions, high-purity water (Epure, Barnstead Co., Newton, MA, USA) was used. The synthetic oligonucleotides, (dT) 8 (mass 2371.59), (dT) 24 (mas, 7238.71), a 5'-dimethoxytritylated 5-mer (5'-DMTr-ATGCG, mass 1805.42), and a 39-mer of the sequence 5'-TGATGATGAT GCGTGAAGAC AGTAGTTCCC TGACTCTGA (mass, 12061.99), were ordered from Microsynth (Balgach, Switzerland) and used without further purification. The mass spectrometers employed for ESI-MS were a Finnigan MAT TSQ 7000 triple quadrupole mass spectrometer, a Finnigan MAT LCQ quadrupole ion trap mass spectrometer (both from Finnigan MAT, San Jose, CA, USA), and a Finnigan MAT 95S double-focusing sector field mass spectrometer (Finnigan MAT, Bremen, Germany).…”

“…1 Its applications include sizing of nucleic acids 2,3 and detection of mutations by exact mass measurements, 4,5 the analysis of mixtures, 6 and sequencing of oligonucleotides to identify modifications 7 or genetic changes. 8 With electrospray ionization (ESI), 9 a process that produces multiply charged intact ions from aqueous solutions of polynucleotides upon ionization of several phosphodiester groups, the mass spectrometric investigation of nucleic acids with molecular masses up to several million dalton is feasible even utilizing mass analyzers that are limited to several thousand m/z units. 10,11 Linear quadrupole (Q), 12 quadrupole ion-trap (QIT), 13 time-of-flight (TOF), 14 double-focusing sector field (DFSF) 15 and Fourier-transform ion cyclotron resonance (FTICR) 16 mass analyzers have been successfully combined with ESI to examine nucleic acids.…”