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PubMed2001

Quantitative detection of Escherichia coli O157 in surface waters by using immunomagnetic electrochemiluminescence.

A protocol for the quantitative detection of Escherichia coli O157 in raw and concentrated surface waters using immunomagnetic electrochemiluminescence (IM-ECL) was developed and optimized. Three antibody sandwich formats were tested: commercial anti-O157:H7 IM beads, IM beads made in-house with a polyclonal anti-O157:H7 immunoglobulin G (IgG), or IM beads made in-house with a monoclonal anti-O157:H7 IgG coupled with a polyclonal anti-O157:H7 IgG to which an electrochemiluminescent label (TAG) was attached. The monoclonal IM bead-polyclonal TAG format was chosen for optimization because it gave lower background levels and linear regression slopes of ca. 1.0, indicative of a constant ECL signal per cell. The dynamic range was ca. 10(1) to 10(5) cells ml(-1) in phosphate-buffered saline and in raw water samples. The monoclonal IM beads selectively captured E. coli O157 cells in the presence of ca. 10(8) cells of a non-O157 strain of E. coli ml(-1). Background ECL signals from concentrated (100-fold) water samples were substantially higher and more variable than raw water samples. The background signal was partially eliminated by the addition of polyvinylpolypyrrolidone. Successive cell capture incubations, termed sequential bead capture (SBC), were optimized for establishing baseline ECL values for individual water samples. The linear dynamic range with SBC was ca. 10(2) to 10(5) E. coli O157 cells ml of concentrated water(-1). To validate the protocol, 10-liter surface water samples were spiked with ca. 5,000 E. coli O157 (Odwalla) cells and concentrated by vortex filtration, and 1- or 3-ml aliquots were analyzed by IM-ECL. Differential ECL signals (SBC) from 1- and 3-ml samples were statistically significant and were generally consistent with standard curves for these cell concentrations. Enrichments were conducted with aliquots of spiked raw water and concentrated water using EC broth and minimal lactose broth (MLB). All tubes with concentrated water became turbid and gave a positive ECL response for E. coli O157 (>10,000 ECL units); MLB gave a somewhat higher detection rate with spiked raw water. The potential sensitivity of the IM-ECL assay is ca. 25 E. coli O157 cells ml of raw water(-1), 25 cells 100 ml of 100-fold concentrated water(-1), or 1 to 2 viable cells liter(-1) with concentration and enrichment. The IM-ECL assay appears suitable for routine analysis and screening of water samples.

Electrochemistry↗

PubMed2003

Low copy number detection of herpes simplex virus type 1 mRNA and mouse Th1 type cytokine mRNAs by Light Cycler quantitative real-time PCR.

The real-time principle of reverse transcriptase polymerase chain reaction (RT-PCR) provides a quantitative and reproducible method to detect low copy number transcripts. The quantitative detection of cytokines from tissue samples is complicated by the low expression rates and the short half-lives of the cytokine proteins. The methods have been insensitive and labor-intensive. The LightCycler technique provides a 30-min PCR system with continuous fluorescent detection, analysis of the melting points of the products and user-friendly software for the analysis of the unknown samples. External copy number standards enable the measurement of amounts of the desired targets. We demonstrate the dynamic range of the RT-PCR system from a 100 to 10(7) mRNA copies of the mouse Th1 cytokines interleukin- (IL-) 12p35, 12p40 and IL-23p19 as well as gamma interferon (IFN-gamma) and the housekeeping gene beta-actin, with the usage of fluorescent hybridization probes. The cytokine quantitation was exemplified in murine nervous system samples. A viral transcript, mRNA of alpha trans-inducing factor (alphaTIF), or VP16 gene, of herpes simplex virus (HSV) type 1 was used to quantitate the viral replication in infected cells and in murine nervous system samples. For this viral transcript the linear dynamic range spanned from ten copies to one million copies (highest tested). For all tested cytokine transcripts, the detection level with the dsDNA binding dye SYBR Green I was one log lower than with the hybridizing fluorescent probes. The viral transcript was detected even with the SYBR Green I system at the level of ten copies. The specificity of the PCR was reached with the use of TaqStart antibody, by careful design of primers and probes, by melting temperature analysis and comparison with the gel electrophoresis and Southern blot analysis.

Animals↗

PubMed1999

Effects of stimulus level on electrode-place discrimination in human subjects with cochlear implants.

Effects of stimulus level on discrimination of one stimulation site from another were examined in 15 human subjects with Nucleus-22 cochlear implant systems. Bipolar stimulation was used in all cases with electrodes in the bipolar pair separated by 1.5 mm (center to center). Subjects were first tested at a medium loudness level, using an adaptive tracking procedure, to determine the regions of the electrode array where electrode-place discrimination was best and the regions where it was poorest. Electrode-place discrimination was then tested at three regions distributed throughout the array, which included the regions of best and poorest discrimination. At each region, electrode-place discrimination was tested at three levels: 25%, 50%, and 75% of the dynamic range. For each of these nine conditions (3 sites x 3 levels), the test-electrode pairs were loudness balanced with the reference-electrode pairs. A two-interval forced-choice same-different procedure was then used to determine discriminability of the reference-electrode pair from the nearest, apical, test-electrode pair. If P(C)max was <0.707 at all three levels, additional testing was done using the next, more apical, electrode pair as the test-electrode pair. A tendency toward better discrimination at more apical regions of the array was observed. Electrode pairs with poor discrimination typically had smaller dynamic ranges than those with good discrimination. There was a weak tendency toward better discrimination at higher levels of stimulation. However, effects of level on electrode-place discrimination were less pronounced and less consistent than previously observed effects of level on temporal discriminations. These results suggest interactions between current spread and the condition of the implanted cochlea as underlying mechanisms.

Adult↗

PubMed2005

Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control.

Quantitation of circulating hepatitis B virus (HBV) DNA is important for monitoring disease progression and for assessing the response to antiviral therapy. Several commercial and 'in house' assays for HBV DNA quantitation have been described but many of these have limitations of relatively low sensitivity and limited dynamic range. This study describes the development and evaluation of a FRET-based real-time PCR assay designed to overcome these limitations and to provide accurate quantitation of DNA from all eight genotypes of HBV (A-H). The assay employs a fully automated nucleic acid extraction system permitting high-sample throughput with minimal 'hands-on' time and incorporates a murine cytomegalovirus (mCMV) internal control to prevent false negative results and under-reporting due to unrecognised problems with viral lysis, DNA purification or PCR amplification. Sensitivity, assessed by Probit analysis at the 95% detection level, was 24.4 IU/ml, associated with an extremely wide dynamic range (approximately 9 log10). Coefficients of variation were low for both intra-assay and inter-assay variability (CV%, 7-11%) and quantitative data correlated well (R2 = 0.97) with the Digene hybrid capture assay. This assay provides an ideal system for therapeutic monitoring and for studying the relationship between HBV viral load and stage of disease.

DNA, Viral↗

PubMed1996

[Use of a digital radiology system with phosphors in the assessment of thoracic diseases in bedside radiography].

We report our experience with a storage phosphor radiography system; to assess both the image resolution and the dynamic range of the system, a phantom simulating chest average density was used. Finally, we investigated the system impact on portable chest radiographs. The radiographs were taken on 35 x 43 cm phosphor plates, with 80 kV, 3 mAs and focus-film distance of 130 cm. The phosphor plates were scanned with a 75 microns laser beam, postprocessed with the system software and, finally, printed on a 20 x 25 cm film. As reported by other Authors, the storage phosphor system showed similar spatial resolution to conventional radiography (2.5 lp/mm for 35 x 43 cm films and 4.5 lp/mm for 20 x 25 cm films), a wide dynamic range always allowing correcting exposure, but increased noise; these features allow film density optimization even in such "critical" situations and bedside radiographs. Pneumonia, pneumothorax, emphysema, pleural effusions and catheters or tubes positioning were always easily depicted. In our experience, the phosphor plate storage system exhibits many advantages over conventional radiography, namely image postprocessing, the opportunity to choose between several sizes of laser films and finally, the possibility of linking the system to a network and possibly to a digital archive. The main drawbacks of the system are its increased noise and, at present, with the available hardware, slow plate reading.

Humans↗

PubMed1983

Tonic-phasic-channel dichotomy and Crozier's law.

The constancy of the dynamic range in a luminance-discrimination task is known as Crozier's law; an old rule says that about half of a log unit spans the range from a low to a high frequency of seeing. For our conditions the slope of the psychometric function is steeper for short than for long test flashes; Crozier's law requires a different constant when temporal parameters change. This result is substantiated by an analysis of Massof's data [Vision Res. 21,995 (1981)] on the variation of the slope of psychometric curves for different wavelengths. The change in Crozier's constant between conditions may reflect the presence of more than one detection channel. If short test flashes are detected by phasic channels and long test flashes by both phasic and tonic channels, then our result implies a shorter dynamic range for phasic (Y-cell) than for tonic (X-cell) channels.

Computers↗

PubMed1998

Comparative study of different standardization concepts in quantitative competitive reverse transcription-PCR assays.

Four different standardization approaches based on a competitive reverse transcription (RT)-PCR assay were compared with a noncompetitive assay based on an external standard curve. Criteria for assessment were accuracy in quantitation, correctness of recovery, sensitivity, dynamic range, reproducibility, throughput, and convenience of sample handling. As a model system, we used the 5'-noncoding region of hepatitis C virus (HCV) for amplification in all quantitative RT-PCRs. A computer program that allowed parallel data processing was developed. Surprisingly, all methods were found suitable for accurate quantitation and comparable with respect to the criterion correctness of recovery. All results differed only by a factor of about 2. The reason for this finding might be that all of our mimics, as well as the wild-type genome of HCV, exhibited exactly the same amplification and hybridization efficacy. Moreover, minimal competition occurred in our experiments over a 5-log dynamic range. A further topic of our investigation was the comparison of two different competitive RNA fragments, mimics, with regard to their suitability as internal standards. One was a heterologous mimic, in which only the primer binding sites were identical to the wild type. The second one was a homologous mimic identical to the wild type except for a small region used for differential hybridization, which was replaced by a permutated sequence of the same length. Both the homologous and heterologous internal mimics were found appropriate for an accurate competitive RT-PCR assay, provided that amplification efficacy, as well as capture efficacy, is proven identical for both analyte and mimic.

Gene Amplification↗

PubMed2003

Multicenter evaluation of the performance characteristics of the NucliSens HIV-1 QT assay used for quantitation of human immunodeficiency virus type 1 RNA.

The analytical performance of the NucliSens HIV-1 QT assay, a highly sensitive test based on nucleic acid sequence-based amplification technology, was evaluated in a multicenter trial. Assay specificity was evaluated with 502 plasma (EDTA) specimens from human immunodeficiency virus type 1 (HIV-1)-seronegative volunteer donors. No HIV-1 RNA was reported in any of the donor specimens. Analytical sensitivity and reproducibility were estimated with panels prepared from a high-titer well-characterized HIV-1 RNA stock (5.84 x 10(8) RNA copies/ml). The assay's dynamic range was linear from 10(6) to 10(1) HIV-1 RNA copies, with a lower detectable limit of 25 copies/ml and a 95% detection rate of 176 copies/ml. Sensitivity of the assay to detect HIV-1 RNA in clinical specimens from patients (n = 101) and in commercially available or prepared panels (n = 24) was compared with NASBA HIV-1 RNA QT (an earlier version of NucliSens HIV-1 QT) and with the Food and Drug Administration-approved standard and ultrasensitive AMPLICOR HIV-1 MONITOR, version 1.0, assays. Detection of HIV-1 RNA was reproducible over a 5-log range (mean standard deviation = 0.15 log). The NucliSens and the standard AMPLICOR assays were equivalent in detection of HIV-1 RNA (concentration, 10(3) to 10(5) copies/ml) in 57 clinical specimens. The NucliSens assay was more sensitive in detecting HIV-1 RNA at lower concentrations (</=10(2) copies/ml) (44 of 44) than either the standard AMPLICOR test (12 of 19) or the NASBA assay (10 of 25). A 25% increase in HIV-1 RNA detection frequency with panels was observed with the NucliSens assay (23 of 24) compared with the standard AMPLICOR test (17 of 24). The new assay was highly specific and demonstrated good sensitivity with a broad linear dynamic range.

HIV-1↗

PubMed2005

Short-term biomarkers of cigarette smoke condensate tumor promoting potential in mouse skin.

Previous studies demonstrated that cigarette smoke condensates (CSCs) possessing significantly different tumorigenic potentials according to a standardized 30-week mouse skin tumor-promotion protocol could likewise be discriminated utilizing short-term indices of sustained hyperplasia and/or inflammation (G. M. Curtin et al., 2004, Toxicol. Sci. 81, 14-25). The current study employed a truncated initiation-promotion protocol to further evaluate CSC-induced hyperplasia, examining issues related to time course of induction, existence of a threshold and suitable dynamic range for detectable responses, and reversibility. Condensate application (9-36 mg "tar"/200-microl application, thrice-weekly for 3-15 weeks) induced treatment-related increases for epidermal thickness, proliferative index as assessed by 5-bromo-2'-deoxyuridine (BrdU) labeling, and ornithine decarboxylase (ODC) expression. Interestingly, observed increases for interfollicular BrdU labeling and ODC expression were partially reversed but still elevated upon cessation of promotion, while increases within the perifollicular epidermis remained elevated at a level similar to that observed during CSC application. In particular, assessments based on perifollicular ODC expression would appear to provide a greater opportunity for test article discrimination based on a rapid time to induction, a low threshold and expanded dynamic range of responses, and the potential to account for irreversible changes. These findings are particularly intriguing based on reports suggesting that ODC expression may be necessary for tumor promotion and that mouse skin tumors originate primarily within the perifollicular epidermis.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1998

Non-linear signal processing in digital hearing aids.

Three different non-linear digital signal processing algorithms were developed; LinEar, DynEar and RangeEar. All three provided individual frequency shaping via a seven-band low-power filterbank and compression in two channels. RangeEar and DynEar used wide dynamic range syllabic compression in the low-frequency (LF) channel, while LinEar used compression limiting. In the high-frequency (HF) channel, RangeEar used a slow-acting automatic volume control, while DynEar and LinEar used compression limiting. Wearable digital signal processing-based experimental instruments were used to evaluate the fitting algorithms under real world conditions with experienced hearing aid users. Evaluation included laboratory testing of speech recognition in noise and questionnaires on sound quality ratings. Results did not indicate one general good-for-all algorithm, but different algorithms resulting in preference and performance depending on the hearing loss configuration. Preference for any of the new algorithms could be predicted based on auditory dynamic range measurements. It was hypothesized that the different preferences were affected by different susceptibility to masking of HF sounds by amplified LF sounds.

Adult↗

PubMed2001

Schemes for the optimization of chest radiography using a computer model of the patient and x-ray imaging system.

A computer program has been developed to model chest radiography. It incorporates a voxel phantom of an adult and includes antiscatter grid, radiographic screen, and film. Image quality is quantified by calculating the contrast (deltaOD) and the ideal observer signal-to-noise ratio (SNR(I)) for a number of relevant anatomical details at various positions in the anatomy. Detector noise and system unsharpness are modeled and their influence on image quality is considered. A measure of useful dynamic range is computed and defined as the fraction of the image that is reproduced at an optical density such that the film gradient exceeds a preset value. The effective dose is used as a measure of the radiation risk for the patient. A novel approach to patient dose and image quality optimization has been developed and implemented. It is based on a reference system acknowledged to yield acceptable image quality in a clinical trial. Two optimizations schemes have been studied, the first including the contrast of vessels as measure of image quality and the second scheme using also the signal-to-noise ratio of calcifications. Both schemes make use of our measure of useful dynamic range as a key quantity. A large variety of imaging conditions was simulated by varying the tube voltage, antiscatter device, screen-film system, and maximum optical density in the computed image. It was found that the optical density is crucial in screen-film chest radiography. Significant dose savings (30%-50%) can be accomplished without sacrificing image quality by using low-atomic-number grids with a low grid ratio or an air gap and more sensitive screen-film system. Dose-efficient configurations proposed by the model agree well with the example of good radiographic technique suggested by the European Commission.

Dose-Response Relationship, Radiation↗

PubMed2000

Electrochemical oxidation of histamine and serotonin at highly boron-doped diamond electrodes.

The electrochemistry of histamine and serotonin in neutral aqueous media (pH 7.2) was investigated using polycrystalline, boron-doped diamond thin-film electrodes. Cyclic voltammetry, hydrodynamic voltammetry, and flow injection analysis (FIA) with amperometric detection were used to study the oxidation reactions. Comparison experiments were carried out using polished glassy carbon (GC) electrodes. At diamond electrodes, highly reproducible and well-defined cyclic voltammograms were obtained for histamine with a peak potential at 1.40 V vs SCE. The voltammetric signal-to-background ratios obtained at diamond were 1 order of magnitude higher than those obtained for GC electrodes at and above 100 microM analyte concentrations. A linear dynamic range of 3-4 orders of magnitude and a detection limit of 1 microM were observed in the voltammetric measurements. Well-defined sweep rate-dependent voltammograms were also obtained for 5-hydroxytryptamine (5-HT). The characteristics of the voltammogram indicated lack of adsorption of its oxidation products on the surface. No fouling or deactivation of the electrode was observed within the experimental time of several hours. A detection limit of 0.5 microM (signal-to-noise ratio 13.8) for histamine was obtained by use of the FIA technique with a diamond electrode. A remarkably low detection limit (10 nM) was obtained for 5-HT on diamond by the same method. Diamond electrodes exhibited a linear dynamic range from 10 nM to 100 microM for 5-HT determination and a range of 0.5-100 microM for histamine determination. The FIA response was very reproducible from film to film, and the response variability was below 7% at the actual detection limits.

Boron↗

PubMed2003

Response growth with sound level in auditory-nerve fibers after noise-induced hearing loss.

People with sensorineural hearing loss are often constrained by a reduced acoustic dynamic range associated with loudness recruitment; however, the neural correlates of loudness and recruitment are still not well understood. The growth of auditory-nerve (AN) activity with sound level was compared in normal-hearing cats and in cats with a noise-induced hearing loss to test the hypothesis that AN-fiber rate-level functions are steeper in impaired ears. Stimuli included best-frequency and fixed-frequency tones, broadband noise, and a brief speech token. Three types of impaired responses were observed. 1) Fibers with rate-level functions that were similar across all stimuli typically had broad tuning, consistent with outer-hair-cell (OHC) damage. 2) Fibers with a wide dynamic range and shallow slope above threshold often retained sharp tuning, consistent with primarily inner-hair-cell (IHC) damage. 3) Fibers with very steep rate-level functions for all stimuli had thresholds above approximately 80 dB SPL and very broad tuning, consistent with severe IHC and OHC damage. Impaired rate-level slopes were on average shallower than normal for tones, and were steeper in only limited conditions. There was less variation in rate-level slopes across stimuli in impaired fibers, presumably attributable to the lack of suppression-induced reductions in slopes for complex stimuli relative to BF-tone slopes. Sloping saturation was observed less often in impaired fibers. These results illustrate that AN fibers do not provide a simple representation of the basilar-membrane I/O function and suggest that both OHC and IHC damage can affect AN response growth.

Acoustic Stimulation↗

PubMed2002

Subjective measures of hearing aid benefit in the NIDCD/VA Clinical Trial.

OBJECTIVE: Subjective measures of performance were assessed on three different hearing aid circuits as part of a large clinical trial. These measurements included the Profile of Hearing Aid Performance and a subjective ranking of individual preference. DESIGN: A multi-center, double-masked clinical trial of hearing aids was conducted at eight VA Medical Centers. Three hearing aid circuits, a linear peak-clipper, a linear compression limiter and a wide dynamic range compressor, were investigated. The experimental design was a three-period, three-treatment crossover design. Subjects (N = 360) were stratified by site and randomized to one of six sequences for the hearing aid circuits. All fittings were binaural and involved a 3-mo trial with each of the three circuits. All subjective measures were administered for unaided and aided conditions at the end of each trial period. RESULTS: While all of the circuits resulted in improved scores on the aided versus the unaided PHAP, there were few conditions in which one circuit outperformed the others. An exception was the aversiveness of sound subscale where the peak clipper frequently scored worse than either the compression limiter or the wide dynamic range compressor. In the subjective ranking scale the compression limiter received more first place rankings than the other two circuits, especially for one subgroup of patients with moderate flat hearing loss. CONCLUSIONS: All circuits were perceived as beneficial by these subjects in most situations. The peak clipper scored worse on aversiveness of sound than did the other two circuits for most subjects, while the compression limiter seemed to have a slight advantage in subjective rankings. Most subjects perceived considerable aided benefit in situations involving background noise and reverberation, situations where hearing aid benefit is often questioned.

Cross-Over Studies↗

PubMed1996

Auditory filter asymmetry at 2000 Hz in 80 normal-hearing ears.

Auditory filter asymmetry, bandwidth, dynamic range, and efficiency was measured at 2000 Hz in both ears of 40 naive, normal-hearing adults, counterbalanced for sex and initial ear tested. The filters were measured with 200-ms tonal signals temporally centered in 300-ms maskers. The maskers were notched noises presented at a spectrum level of 40 dB SPL. Quiet thresholds for the signal were also obtained. The distributions of the measured variables and correlations between them are reported. The average filter was nearly symmetric but the most common filters had steeper upper slopes. The shapes of the two sides of the filter were independent. Filter asymmetry was not correlated with the equivalent rectangular bandwidth (ERB), and quiet threshold was not consistently correlated with the dynamic range of the filter or with ERB. The mean ERB was 255 Hz.

Adolescent↗

PubMed2002

Characterisation of the phosgene response of a membrane inlet 63Ni ion mobility spectrometer.

A membrane inlet 63Ni ion mobility spectrometer interfaced to a quadrupole mass spectrometer with permeation, exponential dilution approaches and syringe-based systems were used to characterise the ion mobility spectrometry (IMS) response to phosgene in dry air (water concentration less than 16.5 mg m(-3)). Phosgene produced a principle product ion in the negative mode with a reduced mobility of 2.16 cm2 V(-1) s(-1), with an unresolved artefact at higher concentrations having a reduced mobility of 2.32 cm2 V(-1) s(-1). The limit of detection of the system with a membrane inlet fitted was estimated to be less than 1 mg m(-3), with an upper limit to the dynamic range of 32 mg m(-3). Mass spectrometric data indicated the existence of [(H2O)nCl]-, [(H2O)nCl2]-; [(H2O)n(O2)Cl]-; [(H2O)n(O)Cl]-; and, [(H2O)n(CO2)Cl]-. The existence of two possible mechanisms for product ion formation is proposed: dissociative electron capture, as well as hydrolysis followed by electron capture. The effect of water contamination of the drying media within the ion mobility spectrometer was also investigated, and the effects were similar to those observed previously with studies on chlorine. Reduced mobility of the product ions was observed to decrease with increasing water contamination of the drying media used within the instrument, while limits of detection increased slightly to less than 2.4 mg m(-3), with no significant effect on dynamic ranges of response or resolution. Preliminary results also indicated a positive mode response for phosgene, albeit at significantly higher concentrations to those observed in the negative mode.

Chemical Warfare Agents↗

PubMed2003

Alterations in dorsal horn neurones in a rat model of cancer-induced bone pain.

Cancer-induced bone pain is a major clinical problem. A rat model based on intra-tibial injection of MRMT-1 mammary tumour cells was used to mimic progressive cancer-induced bone pain. At the time of stable behavioural changes (decreased thresholds to mechanical and cold stimuli) and bone destruction, in vivo electrophysiology was used to characterize natural (mechanical, thermal, and cold) and electrical-evoked responses of superficial and deep dorsal horn neurones in halothane-anaesthetized rats. Receptive field size was significantly enlarged for superficial neurones in the MRMT-1 animals. Superficial cells were characterised as either nociceptive specific (NS) or wide dynamic range (WDR). The ratio of WDR to NS cells was substantially different between sham operated (growth media alone) (26:74%) and MRMT-1 injected rats (47:53%). NS cells showed no significant difference in their neuronal responses in MRMT-1-injected compared to sham rats. However, superficial WDR neurones in MRMT-1-injected rats had significantly increased responses to mechanical, thermal and electrical (A beta-, C fibre-, and post-discharge evoked response) stimuli. Deep WDR neurones showed less pronounced changes to the superficial dorsal horn, however, the response to thermal and electrical stimuli, but not mechanical, were significantly increased in the MRMT-1-injected rats. In conclusion, the spinal cord is significantly hyperexcitable with previously superficial NS cells becoming responsive to wide-dynamic range stimuli possibly driving this plasticity via ascending and descending facilitatory pathways. The alterations in superficial dorsal horn neurones have not been reported in neuropathy or inflammation adding to the evidence for cancer-induced bone pain reflecting a unique pain state.

Action Potentials↗

PubMed2004

Ultrasensitive proteomics using high-efficiency on-line micro-SPE-nanoLC-nanoESI MS and MS/MS.

Ultrasensitive nanoscale proteomics approaches for characterizing proteins from complex proteomic samples of <50 ng of total mass are described. Protein identifications from 0.5 pg of whole proteome extracts were enabled by ultrahigh sensitivity (<75 zmol for individual proteins) achieved using high-efficiency (peak capacities of approximately 10(3)) 15-microm-i.d. capillary liquid chromatography separations (i.e., using nanoLC, approximately 20 nL/min mobile-phase flow rate at the optimal linear velocity of approximately 0.2 cm/s) coupled on-line with a micro-solid-phase sample extraction and a nanoscale electrospray ionization interface to a 11.4-T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS). Proteome measurement coverage improved as sample size was increased from as little as 0.5 pg of sample. It was found that a 2.5-ng sample provided 14% coverage of all annotated open reading frames for the microorganism Deinococcus radiodurans, consistent with previous results for a specific culture condition. The estimated detection dynamic range for detected proteins was 10(5)-10(6). An improved accurate mass and LC elution time two-dimensional data analysis methodology, used to both speed and increase the confidence of peptide/protein identifications, enabled identification of 872 proteins/run from a single 3-h nanoLC/FTICR MS analysis. The low-zeptomole-level sensitivity provides a basis for extending proteomics studies to smaller cell populations and potentially to a single mammalian cell. Application with ion trap MS/MS instrumentation allowed protein identification from 50 pg (total mass) of proteomic samples (i.e., approximately 100 times larger than FTICR MS), corresponding to a sensitivity of approximately 7 amol for individual proteins. Compared with single-stage FTICR measurements, ion trap MS/MS provided a much lower proteome measurement coverage and dynamic range for a given analysis time and sample quantity.

Amino Acid Sequence↗