Daniel P. Little

193 nm photodissociation of larger multiply-charged biomolecules

Abstract In contrast to most ion dissociation methods, 193 nm ultraviolet photodissociation of electrosprayed melittin (2.8 kDa) and ubiquitin (8.6 kDa) molecular ions yields new c and z ions (backbone amine bond dissociation) that provide additional sequence information. Dissociation by collisions or infrared photons produce b and y ions; for cleavages between the same amino acids the c ion represents the addition of NH 2 to the b ion, and z the loss of NH 2 from the y ion, so that these ions can be differentiated by this ± 16.02 Da difference. However, 193 nm photodissociation of 12–29 kDa ions as yet does not give collectable product ions, and that of the very stable y 18 2+ ion from ubiquitin only effects a side chain loss. 193 nm irradiation of negative ions of all-T 30-mer DNA appears to eject electrons; apparently this is the first observation of electron photodetachment from multiply-charged negative ions.

Detection of RET proto-oncogene codon 634 mutations using mass spectrometry

Abstract Mutations located in the RET proto-oncogene at codon 634 associated with multiple endocrine neoplasia type 2A and medullary thyroid carcinoma are detected by low-resolution and high-resolution mass spectrometry schemes not requiring labeling or electrophoretic separation of diagnostic products. The former requires measurement by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of 21- to 27-mer oligonucleotides generated by a primer oligo base extension reaction. The latter is based upon direct measurement of artificial products which include the mutation site using matrix-assisted laser desorption ionization Fourier transform mass spectrometry. In this feasibility study a synthetic 25-mer representing the wildtype allele (7660.3 Da) was easily distinguished from G to A (7644.3 Da) and G to T (7635.3 Da) mutant alleles; the mutant alleles, which differed in mass by only 9.0 Da, were easily resolved when analyzed as a mixture. The results of both detection schemes were highly accurate and reliable, indicating mass spectrometry to be a high-quality alternative for future DNA diagnostics performed in clinical laboratories and genetic profiling studies.

Accurate base composition of double-strand DNA by mass spectrometry

An accurate molecular weight (Mr) assignment for a double-strand (ds) DNA determines or greatly restricts the possible number of each of its four bases, while the compositions for its two single-strand (ss) components can also be derived from their Mr values. For a ds 64-mer (39 kDa), the ss-Mr values (±0.5 Da) of its high-resolution mass spectrum from an electrospray ionization/Fourier transform instrument yield only the correct ds- and ss-base compositions. Literature mass spectra of lower mass accuracy show that such data can also restrict their possible composition assignments, with further discrimination using the abundance vs. base composition of small fragment ions from the dissociation of the ss molecular ions.

Estimation of DNA sequence diversity in bovine cytokine genes

Abstract. DNA sequence variation provides the fundamental material for improving livestock through selection. In cattle, single nucleotide polymorphisms and small insertions/deletions (collectively referred to here as SNPs) have been identified in cytokine genes and scored in a reference population to determine linkage map positions. The aim of the present study was twofold: first, to estimate the SNP frequency in a reference population of beef cattle, and second, to determine cytokine haplotypes in a group of sires from commercial populations. Forty-five SNP markers in DNA segments from nine cytokine gene loci were analyzed in 26 reference parents. Comparison of all 52 haploid genomes at each PCR amplicon locus revealed an average of one SNP per 143 bp of sequence, whereas comparison of any two chromosomes identified heterozygous sites, on average, every 443 bp. The combination of these 45 SNP genotypes was sufficient to uniquely identify each of the 26 animals. The average number of haplotype alleles (4.4) per PCR amplicon (688 bp) and the percentage heterozygosity among founding parents (50%) were similar to those for microsatellite markers in the same population. For 49 sires from seven common breeds of beef cattle, SNP genotypes (1225 total) were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) at three amplicon loci. All three of the amplicon haplotypes were correctly deduced for each sire without the use of parent or progeny genotypes. The latter allows a wide range of genetic studies in commercial populations of cattle where genotypic information from relatives may not be available.

Direct detection of synthetic and biologically generated double-stranded DNA by MALDI-TOF MS

Abstract A synthetic 50-mer DNA was mixed at room temperature with a non-complementary or a complementary 27-mer and analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. The former resulted in negligible signals from a 27-/50- mer heterodimer, for the latter this was a dominant peak, consistent with maintaining Watson-Crick interactions in the gas phase. Double stranded DNA derived from an enzymatic digestion of a 252-base-pair polymerase chain reaction product when prepared at room temperature yielded very little specific double stranded DNA, but sample preparation at reduced temperature resulted in spectra dominated by the expected heterodimer signals with no random (i.e. non-Watson-Crick) dimers. As a DNA diagnostics genotyping application, apolipoprotein-E alleles were easily distinguished considering only the masses of their double stranded DNA digest fragments.

INFRARED PHOTODISSOCIATION OF NON-COVALENT ADDUCTS OF ELECTROSPRAYED NUCLEOTIDE IONS

Efficient dissociation of gaseous non-covalent adducts of multiply charged DNA anions can be effected by infrared irradiation to yield minimal dissociation of the DNA molecular ions-far less dissociation than by collisional activation. Examples include removal of adducted impurities from 100-mer DNA anions and removal of all 14 adducted molecules of 1,4-diaminobutane from M15− to M17− of a 50-mer DNA.

DNA sequencing with blackbody infrared radiative dissociation of electrosprayed ions

Abstract As shown by Williams for positive multiply-charged ions, negative electrosprayed ions trapped in a Fourier-transform ion cyclotron resonance mass spectrometer can be dissociated efficiently with BIRD. For a 50-mer DNA, an ion cell wall temperature of 90°C gives no dissociation and 120°C gives only uninformative base loss. However, 150°C gives extensive dissociation producing ionic products qualitatively similar to those from laser IR, collisionally activated, and nozzle-skimmer dissociation. Under the conditions used, BIRD produces more fragment ions that have lost an additional base (AH). Structurally informative BIRD spectra are also observed for a 100-mer DNA and a double-strand 64-mer DNA. For both samples, BIRD successfully ‘boiled off’ non-covalent adducts that produced an uninterpretable spectrum, while laser IR was only successful in this for the ds 64-mer.

Double stranded DNA sequencing by tandem mass spectrometry

Abstract Electrospray ionization produces far more abundant molecular ions for double stranded (ds) DNA than for single stranded (ss) and accurate molecular masses can provide the base composition of dsDNA. This study shows that the Fourier-transform mass spectra of a ds 64-mer DNA can provide approximately 50% more sequence information than the spectra of either individual ss component. Cleavages triggered by T loss are of low probability, but the opposite is true in the complementary ss strand that bears the base A at this site. The spectrum of the product of the attempted biological synthesis of a ds 70-mer DNA provided critical information on its extensive 3′-end heterogeneity.

Dna Analysis by Mass Spectrometry: Applications in Dna Sequencing And Dna Diagnostics

Abstract Use of mass spectrometry to detect PCR amplified DNA from individuals infected with hepatitis B virus, to derive DNA sequence information through exo-nuclease based degradation or Sanger sequencing and a new format (PROBE) for the efficient determination of mutations is described.

Complete large-molecule high-resolution mass spectra from 50-femtomole microvolume injection.

For electrospray ionization in Fourier-transform mass spectrometry, direct injection of 5×10−14 mol (0.5 µL of 100 nM from a microvolume sample valve) of ubiquitin (8565 Da) into the flowing solvent stream yields a spectrum with 85:1 signal-to-noise ratio, 2-ppm mass accuracy, and isotopic resolution. Gated trapping for 100 µs from a 0.15-µL/min injection of 20-µM ubiquitin consumes 5×10−18 mol, which produces a spectrum with 23:1 signal-to-noise ratio and τ;3×105 resolving power.