We describe a new technology for the molecular genotyping of microbes using a platform known commercially as the Ibis T5000. The technology couples multilocus polymerase chain reaction (PCR) to ...electrospray ionization/mass spectrometry (PCR/ESI-MS) and was developed to provide rapid, high-throughput, and precise digital analysis of either isolated colonies or original patient specimens on a platform suitable for use in hospital or reference diagnostic laboratories or public health settings. The PCR/ESI-MS method measures digital molecular signatures from microbes, enabling real-time epidemiological surveillance and outbreak investigation. This technology will facilitate understanding of the pathways by which infectious organisms spread and will enable appropriate interventions on a time frame not previously achievable.
We describe a new approach for infectious disease surveillance that facilitates rapid identification of known and emerging pathogens. The process uses broad-range polymerase chain reaction (PCR) to ...amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry for accurate mass measurements of PCR products, and base composition signature analysis to identify organisms in a sample. We demonstrate this principle by using 14 isolates of 9 diverse Coronavirus spp., including the severe acute respiratory syndrome-associated coronavirus (SARS-CoV). We show that this method could identify and distinguish between SARS and other known CoV, including the human CoV 229E and OC43, individually and in a mixture of all 3 human viruses. The sensitivity of detection, measured by using titered SARS-CoV spiked into human serum, was approximate, equals1 PFU/mL. This approach, applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens, is capable of automated analysis of >900 PCR reactions per day.
Analysis of nucleic acids by FTICR MS Hofstadler, Steven A.; Sannes-Lowery, Kristin A.; Hannis, James C.
Mass spectrometry reviews,
March/April 2005, Letnik:
24, Številka:
2
Journal Article
A dual electrospray ionization (ESI) source employed with hexapole accumulation and gated trapping provides a novel method of using an internal standard to achieve high mass accuracies in Fourier ...transform ion cyclotron resonance mass spectrometry. Two ESI emitters are sequentially positioned in front of the heated metal capillary inlet by a solenoid fitted to an XYZ micromanipulator; one emitter contains the analyte(s) of interest and the other an internal standard. A 5 V transistor–transistor logic pulse from the data station controls the solenoid by means of a solid-state relay so that matching of spectral peak intensities (i.e., analyte and internal standard intensities) can be accomplished by adjusting the hexapole accumulation time for each species. Polythymidine, d(pT)
18, was used as the internal standard for all studies reported here. The absolute average error for an internally calibrated 15-mer oligonucleotide (theoretical monoisotopic mass = 4548.769 Da) was −1.1 ppm (external calibration: 41 ppm) with a standard deviation of ±3.0 ppm (external calibration: ±24 ppm) for a total of 25 spectra obtained at various hexapole accumulation time ratios. Linear least squares regression analysis was carried out and revealed a linear dependence of the magnitudes of the peak height ratios (analyte/internal standard) vs. hexapole accumulation time ratios (analyte/internal standard) which is described by the following equation:
y = 0.45
x −0.02. The fitted line had a %RSD of the slope of 28% with an
R
2 of 0.93. The applicability of this methodology was extended to a polymerase chain reaction product with a theoretical average molecular mass of 50,849.20 Da. With the internal standard, d(pT)
18, an absolute average error of −8.9 ppm (external calibration: 44 ppm) based on five measurements was achieved with a standard deviation of 11 ppm (external calibration: ±36 ppm), thus illustrating this method’s use for characterizing large biomolecules such as those encountered in genomics and proteomics related research.
High-mass accuracy is demonstrated using internal calibration for product ions produced by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) of a 15-mer oligonucleotide, ...5‘-(CTG)5-3‘. Internal calibration for this tandem MS experiment was accomplished using a dual electrospray ionization (ESI) source coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) utilizing hexapole accumulation and gated trapping. The pulse sequence entails injection, trapping, and gas-phase isolation of the precursor ion of interest followed by the SORI-CID of this ion and, subsequently, injection and trapping of the internal mass calibrant (i.e., poly(ethylene glycol) with a 1000 Da average mass). The product ions and the poly(ethylene glycol) ions are then simultaneously excited by a broadband frequency chirp excitation waveform and detected. This technique corrects for space-charge effects on the measurement of an ion's cyclotron frequency experienced when externally calibrated data are used. While external calibration for FTICR-MS can result in mass errors of greater than 100 ppm, this internal standardization method demonstrated significantly more consistent accurate mass measurements with average mass errors ranging from −1.2 to −3.2 ppm for the 15-mer oligonucleotide used in this study. This method requires limited modifications to ESI-FTICR mass spectrometers and is applicable for both positive and negative modes of ionization as well as other sample types (e.g., pharmaceuticals, proteins, etc.).
Epidemiological and forensic analyses of bioterrorism events involving Bacillus anthracis could be improved if both variable number tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs) ...could be combined on a single analysis platform. Here we present the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) to characterize 24 alleles from 6 VNTR loci and 11 alleles from 7 SNP loci in B. anthracis. The results obtained with ESI-FTICR-MS were consistent with independent results obtained from traditional approaches using electrophoretic detection of fluorescent products. However, ESI-FTICR-MS improves on the traditional approaches because it does not require fluorescent labeling of PCR products, minimizes post-PCR processing, obviates electrophoresis, and provides unambiguous base composition of both SNP and VNTR PCR products. In addition, ESI-FTICR-MS allows both marker types to be examined simultaneously and at a rate of approximately 1 sample per min. This technology represents a significant advance in our ability to rapidly characterize B. anthracis isolates using VNTR and SNP loci.
The utility of electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry as a new approach for genotyping short tandem repeats (STRs) is demonstrated. STRs are ...currently valued as a powerful source of genetic information with repeats that range in structure from simple to hypervariable. Two tetranucleotide STR loci were chosen to evaluate ESI-FTICR mass spectrometry as a tool for genotyping: HUMTH01, a simple STR with nonconsensus alleles, and vWA, a compound STR with nonconsensus alleles. For HUMTH01, the genotype (i.e., repeat number of each allele) was determined for each of 30 individuals using mass measurements of double-stranded amplicons. Low-intensity peaks observed in the spectra of amplicons derived from heterozygous individuals were identified by mass as heteroduplexes that had formed between nonhomologous strands. Mass measurement of the double-stranded vWA amplicon was not sufficient for determining whether the individual was homozygous for allele subtype 18 or 18‘ since the amplicons differ by only 0.99 Da. Therefore, single-stranded amplicons were generated by incorporating a phosphorylated primer, prepared using T4 polynucleotide kinase, into the PCR phase and subsequently digesting the bottom strand using λ-exonuclease. Accurate mass measurements were obtained for the single-stranded amplicons using internal calibration and the addition of a correction factor to adjust for the natural variation of isotopic abundances, confirming that the individual is homozygous for allele 18. Our results clearly demonstrate that ESI-FTICR mass spectrometry is a powerful approach to characterize both simple and compound STRs beyond the capabilities of electrophoretic technologies.
In traditional approaches, mitochondrial DNA (mtDNA) variation is exploited for forensic identity testing by sequencing the two hypervariable regions of the human mtDNA control region. To reduce time ...and labor, single nucleotide polymorphism (SNP) assays are being sought to possibly replace sequencing. However, most SNP assays capture only a portion of the total variation within the desired regions, require a priori knowledge of the position of the SNP in the genome, and are generally not quantitative. Furthermore, with mtDNA, the clustering of SNPs complicates the design of SNP extension primers or hybridization probes. This article describes an automated electrospray ionization mass spectrometry method that can detect a number of clustered SNPs within an amplicon without a priori knowledge of specific SNP positions and can do so quantitatively. With this technique, the base composition of a PCR amplicon, less than 140 nucleotides in length, can be calculated. The difference in base composition between two samples indicates the presence of an SNP. Therefore, no post-PCR analytical construct needs to be developed to assess variation within a fragment. Of the 2754 different mtDNA sequences in the public forensic mtDNA database, nearly 90% could be resolved by the assay. The mass spectrometer is well suited to characterize and quantitate heteroplasmic samples or those containing mixtures. This makes possible the interpretation of mtDNA mixtures (as well as mixtures when assaying other SNPs). This assay can be expanded to assess genetic variation in the coding region of the mtDNA genome and can be automated to facilitate analysis of a large number of samples such as those encountered after a mass disaster.
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