James C. Hannis

The Ibis T5000 Universal Biosensor: An Automated Platform for Pathogen Identification and Strain Typing

We describe a new approach to the sensitive and specific identification of bacteria, viruses, fungi, and protozoa based on broad-range PCR and high-performance mass spectrometry. The Ibis T5000 is based on technology developed for the Department of Defense known as T.I.G.E.R. (Triangulation Identification for the Genetic Evaluation of Risks) for pathogen surveillance. The technology uses mass spectrometry—derived base composition signatures obtained from PCR amplification of broadly conserved regions of the pathogen genomes to identify most organisms present in a sample. The process of sample analysis has been automated using a combination of commercially available and custom instrumentation. A software system known as T-Track manages the sample flow, signal analysis, and data interpretation and provides simplified result reports to the user. No specialized expertise is required to use the instrumentation. In addition to pathogen surveillance, the Ibis T5000 is being applied to reducing health care—associated infections (HAIs), emerging and pandemic disease surveillance, human forensics analysis, and pharmaceutical product and food safety, and will be used eventually in human infectious disease diagnosis. In this review, we describe the automated Ibis T5000 instrument and provide examples of how it is used in HAI control.

A dual electrospray ionization source combined with hexapole accumulation to achieve high mass accuracy of biopolymers in fourier transform ion cyclotron resonance mass spectrometry

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 = 0.45x −0.02. The fitted line had a %RSD of the slope of 28% with anR2 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.

Nanoelectrospray mass spectrometry using non‐metalized, tapered (50 → 10 μm) fused‐silica capillaries

Nanoelectrospray emitter tips, pulled from fused-silica capillaries requiring no chemical treatment or metal coating, have been operated using remote coupling of the electrospray voltage. Using common laboratory implements, 50 μm i.d. fused-silica capillaries were pulled to ca. 10 μm, and attached to a stainless steel capillary via a teflon coupler for the production of nanoelectrospray (i.e. nL/min flowrates). Using a variety of different tips, flowrates were determined to range from 30 to 80 nL/min. Spectra of bovine insulin (ca. 6 kDa) and Angiotensin III (ca. 1 kDa) produced signal-to-noise (S/N) ratios of ca.500 and ca.100, respectively; each analysis consuming ca. 10 femtomoles of sample. Radical cation formation, in addition to the monoprotonated molecule was also observed during nanoelectrospray of angiotensin III which implies that this nanoelectrospray source should be applicable to trace amounts of non-polar compounds. The stability of the ion source is demonstrated by measuring the current of the electrosprayed ions at the shutter of the Fourier transform ion cyclotron resonance mass spectrometer as a function of time. Overall stability of the nanoelectrospray for angiotensin III, using no pressure to initialize or stabilize the electrospray, was determined as 23 pA ± 0.97 pA (4.2 % relative standard deviation) over a 30 minute period.

Molecular Genotyping of Microbes by Multilocus PCR and Mass Spectrometry: A New Tool for Hospital Infection Control and Public Health Surveillance

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.

High-Resolution Genotyping of Campylobacter Species by Use of PCR and High-Throughput Mass Spectrometry

ABSTRACT In this work we report on a high-throughput mass spectrometry-based technique for the rapid high-resolution identification of Campylobacter jejuni strain types. This method readily distinguishes C. jejuni from C. coli, has a resolving power comparable to that of multilocus sequence typing (MLST), is applicable to mixtures, and is highly automated. The strain typing approach is based on high-performance mass spectrometry, which “weighs” PCR amplicons with enough mass accuracy to unambiguously determine the base composition of each amplicon (i.e., the numbers of As, Gs, Cs, and Ts). Amplicons are derived from PCR primers which amplify short (<140-bp) regions of the housekeeping genes used by conventional MLST strategies. The results obtained with a challenge panel that comprised 25 strain types of C. jejuni and 25 strain types of C. coli are presented. These samples were parsed and resolved with demonstrated sensitivity down to 10 genomes/PCR from pure isolates.

Characterization of a microdialysis approach to prepare polymerase chain reaction products for electrospray ionization mass spectrometry using on‐line ultraviolet absorbance measurements and inductively coupled plasma‐atomic emission spectroscopy

The desalting efficiencies for microdialysis have been quantitatively determined using inductively coupled plasma atomic emission spectroscopy (ICP-AES) by analyzing samples which mimic polymerase chain reaction (PCR) conditions. Desalting efficiencies for the removal of Mg2+ from samples containing oligonucleotides were typically 99.94% for a single dialysis experiment and 99.98% for a tandem-dialysis experiment. The determination of the molecular weight selectivity of a microdialysis fiber with a 13,000 Da molecular weight cutoff (MWCO) employing a 10 mM NH4OAc countercurrent buffer was accomplished using on-line ultraviolet (UV) absorbance. Results revealed an insensitivity to molecular weight over a broad range represented by dNTPs (ca. 490 Da), PCR primers (ca. 5100 Da), and PCR products (extending to ca. 100 kDa) thus showing the inability of microdialysis to remove unincorporated deoxyribonucleotide 5′-triphosphates (dNTPs) and primers from PCRs. A comparison study conducted utilizing proteins ranging from ca. 1 to 29 kDa produced recoveries dictated by the MWCO of the microdialysis fiber. Recoveries were zero for the smallest proteins tested (ca. 1.3 and 2.9 kDa), but increased to 98% for the largest protein (ca. 29 kDa). The ability of microdialysis to convert double-stranded DNA to single-stranded DNA due to a rapid decrease in the ionic strength was examined along with the effects of buffer concentration, temperature, pH, tandem dialysis, and a denaturing additive, formamide. Melting temperature curves showed that microdialyzed samples remain double stranded while utilizing NH4OAc buffer concentrations of 0 (i.e. pure water) to 10 mM. Adjustment of the buffer up to pH 10.98, temperature increases to 51 °C, tandem dialysis, and the addition of formamide were also unable to produce the conversion of ds-DNA to ss-DNA in detectable amounts. Copyright

Tailoring the gas-phase dissociation and determining the relative energy of activation for dissociation of 7-deaza purine modified oligonucleotides containing a repeating motif

Abstract 7-Deaza purine modified oligonucleotides with a repeating motif have been sequenced by slow heating methods illustrating the ability to alter the preferred unimolecular decomposition pathway. The modified oligonucleotides limited the number of product ions per repeat unit resulting in an increased signal-to-noise ratio for the tandem mass spectrometry data. Loss of 7-deaza purine nucleobases or 7-deaza purine related product ions were not observed. The results illustrate the importance of the N7 position of purine nucleobases for low energy gas-phase decomposition. FRAGMENT results showed that for the 3 − charge state of the 16-mer oligonucleotides, 5′-(AATG) 4 -3′ and 5′-(c 7 Ac 7 ATG) 4 -3′, that the 7-deaza deoxyadenosine modified sequence had a 38% greater relative energy of activation for unimolecular decomposition when compared to the unmodified sequence. For large DNA molecules that have purine-rich repeat units, the multiple sequence sites per repeat can result in a substantial loss of sequence information over large areas due to a reduced signal-to-noise ratio of the product ion spectrum. The incorporation of 7-deaza purines can limit the number of product ions during gas phase sequencing while ensuring sufficient sequence coverage to localize mutations or polymorphisms housed within a specific repeat unit.

Genotyping complex short tandem repeats using electrospray ionization Fourier transform ion cyclotron resonance multistage mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is a rapidly emerging, universal platform with the ability to provide detailed information regarding genetic variation and the up- and down regulation of their cognate gene products. Herein, we report our progress towards the development of ESI-FTICR mass spectrometry for the characterization of genomic regions which contain both a length and sequence polymorphism (i.e., complex short tandem repeats). Specifically, it is demonstrated for the first time that a high-quality ESI-FTICR mass spectrum of a 82-bp double- stranded PCR product derived from a single, 50 (mu) L PCR reaction with less than 10 X 10-15 moles injected into the mass spectrometer can be routinely obtained. It is important to note that each measurement, which translates to an accurate genotype, is completed on the timescale of seconds. Progress towards the implementation of flow- injection analysis methodology to increase the throughput is also presented using an alternating injection of a 15-mer and 16-mer oligonucleotide.