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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.

In this work, we present a simple method by which to preferentially detect either high molecular weight or low molecular weight ions generated by electrospray ionization. This approach, termed selective ion filtering by digital thresholding (SIFdT) is demonstrated on a commercial ESI-TOF instrument that employs a fast digitizer coupled to a microchannel plate detector. The digital representation of each individual scan is digitally filtered prior to spectral coaddition. As larger, more highly charged ions induce a more intense response than low molecular weight singly charged species, a digital threshold can be set that precludes the detection of singly charged species yet permits the efficient detection of larger, more highly charged species. In this work, we demonstrate the applicability of this approach to eliminate low molecular weight chemical noise in ESI-TOF spectra of oligonucleotide and protein ions, demonstrate improved dynamic range for analyte solutions containing high levels of low MW constituents, and show that spectra acquired at different digital thresholds can be subtracted to yield spectra of low molecular weight constituents with improved mass measurement accuracies. A notional scheme is presented in which an alternative digitization approach is employed using multiple differentially thresholded data streams to allow improved internal mass calibration and higher resolution ion partitioning.

A novel scheme for performing infrared multiphoton dissociation (IRMPD) is presented in which a hollow fiber waveguide (HFWG) is used to transmit IR radiation into the ion storage region of a mass spectrometer. Efficient dissociation of oligonucleotide and protein ions is demonstrated on an ESI-FTICR instrument in which IRMPD is performed in the external ion reservoir and on a quadrupole ion trap. Using a simple optical scheme consisting of a single focusing lens and an x, y translator, the 10.6-[micro]m IR laser beam, initially 3.5 mm in diameter, is focused into the vacuum-sealed HFWG. The small internal diameter and the high transfer efficiency of the waveguide allow IR radiation of high power density to be employed for IRMPD. In studies performed on a quadrupole ion trap, a 500-[micro]m-i.d. waveguide was used as a medium to transmit IR radiation directly through a 700-[micro]m orifice in the ring electrode. Efficient IRMPD of both a 12-mer oligonucleotide and the protein melittin were performed at laser powers of 0.5 and 3.2 W, respectively.

Electrospray ionization mass spectrometry was used in the characterization of low-affinity complexes between RNA and small molecules.

The pursuit of ever higher mass-resolving power in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has driven a demand for higher magnetic field strength and longer time-domain ICR signal lifetime, with proportionate increase in data set size in direct-mode detection. Heterodyne-mode detection thus becomes increasingly important for achieving ultrahigh-mass resolution from a fixed maximum-size data set. Unfortunately, as conventionally performed (i.e., analog single phase), heterodyne detection reduces the S/N ratio by a factor of 2(super 1/2) relative to direct-mode detection. Here, we restore the factor of 2(super 1/2) by use of quadrature heterodyne detection. In addition, replacement of analog by digital heterodyning eliminates analog circuitry (and its associated noise). Finally, digital filtering of the timedomain discrete ICR signal not only eliminates the need for a bank of analog low-pass filters but also ensures that the Nyquist bandwidth and filter bandwidth are always matched, for optimal noise reduction. With these features, digital quadrature heterodyne detection becomes the detection method of choice for high-resolution FT-ICR MS.

A single population of multiply charged protein ions formed by electrospray ionization is held in a trapped ion cell and remeasured continuously by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry while undergoing multiple reactions with diethylamine. In a first example, electrosprayed horse myoglobin with an average of 16 attached protons is reacted with base at a pressure of 1.5 X [10.sup.8] Torr for a period of 60 s. A total of 31 spectra acquired with a duty cycle of 2 s exhibit the charge state-dependent formation of up to three diethylamine adducts and removal of up to nine protons. A real-time measuring experiment is then conducted over a 1 h period to observe charge stripping of electrosprayed myoglobin ions, leaving as few as seven charges. Real-time monitoring is used to evaluate the effect of reagent gas pressure on adduct formation and is used in conjunction with high mass resolution FTICR detection to resolve the isotopic peaks within individual charge states of adduct spectra. The attractive features of real-time reaction monitoring include a dramatic reduction in experiment time and sample consumed while concurrently observing long-lived intermediates and reaction products as they form.

Presents information about a novel scheme for performing infrared multiphoton dissociation (IRMPD) in which a hallow fiber waveguide (HFWG) is used to transmit IR radiation into the ion storage region of a mass spectrometer. Account of materials and methods used to develop this scheme; Schematic representation of the HFWG-IRMPD coupling approach used in this work; Overview of results of IRMPD in the external ion reservoir of an electrospray ionization-fourier transform ion cyclotron resonance mass spectrometry; Account of HFWG-IRMPD in an external ion reservoir; Comparison of IRMPD with oligonucleotides on the basis of power required for efficient dissociation.

Describes an improved, high-performance version of the concentric vacuum chamber shown for forming ions at high pressure in a strong magnetic field and detecting them in an adjacent Fourier transform ion cyclotron resonance mass spectrometry trapped ion cell. Addition of a mechanical shutter to halt the flow of neutrals to the trapped ion cell.

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.