Orders Of Magnitude Dynamic Range Research Articles

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry for quantitative parallel reaction monitoring of peptide abundance and single-shot proteomic analysis of a human cell line

We coupled capillary zone electrophoresis (CZE) with an ultrasensitive electrokinetically pumped nanospray ionization source for tandem mass spectrometry (MS/MS) analysis of complex proteomes. We first used the system for the parallel reaction monitoring (PRM) analysis of angiotensin II spiked in 0.45mg/mL of bovine serum albumin (BSA) digest. A calibration curve was generated between the loading amount of angiotensin II and intensity of angiotensin II fragment ions. CZE-PRM generated a linear calibration curve across over 4.5 orders of magnitude dynamic range corresponding to angiotensin II loading amount from 2amole to 150fmole. The relative standard deviations (RSDs) of migration time were <4% and the RSDs of fragment ion intensity were ∼20% or less except 150fmole angiotensin II loading amount data (∼36% RSD). We further applied the system for the first bottom up proteomic analysis of a human cell line using CZE-MS/MS. We generated 283 protein identifications from a 1h long, single-shot CZE MS/MS analysis of the MCF7 breast cancer cell line digest, corresponding to ∼80ng loading amount. The MCF7 digest was fractionated using a C18 solid phase extraction column; single-shot analysis of a single fraction resulted in 468 protein identifications, which is by far the largest number of protein identifications reported for a mammalian proteomic sample using CZE.

Subpicogram Per Milliliter Detection of Interleukins Using Silicon Photonic Microring Resonators and an Enzymatic Signal Enhancement Strategy

The detection of biomolecules at ultralow (low to subpicogram per milliliter) concentrations and within complex, clinically relevant matrices is a formidable challenge that is complicated by limitations imposed by the Langmuir binding isotherm and mass transport, for surface-based affinity biosensors. Here we report the integration of an enzymatic signal enhancement scheme onto a multiplexable silicon photonic microring resonator detection platform. To demonstrate the analytical value of this combination, we simultaneously quantitated levels of the interleukins IL-2, IL-6, and IL-8 in undiluted cerebrospinal fluid in an assay format that is multiplexable, relatively rapid (90 min), and features a 3 order of magnitude dynamic range and a limit of detection ≤1 pg/mL. The modular nature of this assay and technology should lend itself broadly amenable to different analyte classes, making it a versatile tool for biomarker analysis in clinically relevant settings.

Integration of On-Chip Isotachophoresis and Functionalized Hydrogels for Enhanced-Sensitivity Nucleic Acid Detection

We introduce an on-chip electrokinetic assay to perform high-sensitivity nucleic acid (NA) detection. This assay integrates electrokinetic sample focusing using isotachophoresis (ITP) with a background signal-removal strategy that employs photopatterened, DNA-functionalized hydrogels. In this multistage assay, ITP first enhances hybridization kinetics between target NAs and end-labeled complementary reporters. After enhanced hybridization, migration through a DNA-functionalized hydrogel region removes excess reporters through affinity interactions. We demonstrate our assay on microRNAs, an important class of low-abundance biomarkers. The assay exhibits 4 orders of magnitude dynamic range, near 1 pM detection limits starting from less than 100 fg of microRNA, and high selectivity for mature microRNA sequences, all within a 10 min run time. This new microfluidic framework provides a unique quantitative assay for NA detection.

ESTIMATING THE STAR FORMATION RATE AT 1 kpc SCALES IN NEARBY GALAXIES

Using combinations of H\alpha, ultraviolet (UV), and infrared (IR) emission, we estimate the star formation rate (SFR) surface density, \Sigma_SFR, at 1 kpc resolution for 30 disk galaxies that are targets of the IRAM HERACLES CO survey. We present a new physically-motivated IR spectral energy distribution-based approach to account for possible contributions to 24\mum emission not associated with recent star formation. Considering a variety of "reference" SFRs from the literature, we revisit the calibration of the 24\mum term in hybrid (UV+IR or H\alpha+IR) tracers. We show that the overall calibration of this term remains uncertain at the factor of two level because of the lack of wide-field, robust reference SFR estimates. Within this uncertainty, published calibrations represent a reasonable starting point for 1 kpc-wide areas of star-forming disk galaxies but we re-derive and refine the calibration of the IR term in these tracers to match our resolution and approach to 24\mum emission. We compare a large suite of \Sigma_SFR estimates and find that above \Sigma_SFR \sim 10^-3 M_\odot yr^-1 kpc^-2 the systematic differences among tracers are less than a factor of two across two orders of magnitude dynamic range. We caution that methodology and data both become serious issues below this level. We note from simple model considerations that focusing on a part of a galaxy dominated by a single stellar population the intrinsic uncertainty in H\alpha and FUV-based SFRs are \sim 0.3 and \sim 0.5 dex.

Metabolic Cytometry: Capillary Electrophoresis with Two-Color Fluorescence Detection for the Simultaneous Study of Two Glycosphingolipid Metabolic Pathways in Single Primary Neurons

Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least 6 orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.

DNA microarray fabricated on poly(acrylic acid) brushes-coated porous silicon by in situ rolling circle amplification

Microarrays hold considerable promise in large-scale biology on account of their analytical, massive and parallel nature. In a step toward further enabling such a capability, we describe the application of rolling circle amplification (RCA) for a sensitive and multiplex detection of nucleic acid targets on oligonucleotide-conjugated polymer brushes covalently grown from porous silicon. Both RCA and polymer brushes have been taken to increase the loading quantity of target molecules and thus improve the detection sensitivity without loss of multiplexing. Besides, polymer brushes were employed to protect porous silicon and to provide biologically simulated environments, making the attached biomolecules maintain bioactivity. This approach can reach a detection limit of 0.1 nM target analytes and three orders of magnitude dynamic range of 0.1-100 nM, with a fluorescence scanner. A two-colour DNA microarray was achieved via RCA of two kinds of circular DNA targets on one chip simultaneously. The porous silicon chip-based RCA technique is promising for the multiplex detection of deoxynucleic acids on microarrays.

Ultra High Resolution Linear Ion Trap Orbitrap Mass Spectrometer (Orbitrap Elite) Facilitates Top Down LC MS/MS and Versatile Peptide Fragmentation Modes

Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm—incorporating phase information—further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s—increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.

A microfluidic based differential plasmon resonance sensor

A new type of differential surface plasmon (SPR) sensor integrated with a microfluidic system is presented. The working principle of the microfluidic device is based on hydrodynamic modulation of two laminar streams inside a microchannel to provide periodic changes of the environment on the SPR sensor. The modulated reflectance is then demodulated using a lock-in amplifier. The presented sensor provides sensitivities of index of refraction about 4 × 10 −8 RIU together with a 4 orders of magnitude dynamic range. This method demonstrates a sensitive detection scheme which could be used for label-free detection.

Nine orders of magnitude dynamic range: picomolar to millimolar concentration measurement in capillary electrophoresis with laser induced fluorescence detection employing cascaded avalanche photodiode photon counters.

The dynamic range of capillary electrophoresis analysis is ultimately limited by molecular shot noise at low concentrations and by concentration-induced band broadening at high concentrations. We report a system that approaches these fundamental limits. A laser-induced fluorescence detector is reported that employs a cascade of four fiber-optic beam splitters connected in series to generate a primary signal and four attenuated signals, each monitored by a single-photon counting avalanche photodiode. Appropriate scaling of the signals from the five photodiodes produces a linear optical calibration curve for 5-carboxyl-tetramethylrhodamine from the concentration detection limit of 1 pM to the upper limit of 1 mM. Mass detection limits are 120 yoctomoles (70 molecules) injected into the instrument. The very-wide dynamic range instrument was used to study the metabolic products of the fluorescently labeled glycosphingolipid tetramethylrhodamine labeled GM1 (GM1-TMR) produced by single cells isolated from the rat cerebellum.

Real-time cancellation of temperature induced resonance shifts in SOI wire waveguide ring resonator label-free biosensor arrays

A comprehensive investigation of real-time temperature-induced resonance shift cancellation for silicon wire based biosensor arrays is reported for the first time. A reference resonator, protected by either a SU8 or SiO(2) cladding layer, is used to track temperature changes. The temperature dependence of resonators in aqueous solutions, pertinent to biosensing applications, is measured under steady-state conditions and the operating parameters influencing these properties are discussed. Real-time measurements show that the reference resonator resonances reflect the temperature changes without noticeable time delay, enabling effective cancellation of temperature-induced shifts. Binding between complementary IgG protein pairs is monitored over 4 orders of magnitude dynamic range down to a concentration of 20 pM, demonstrating a resolvable mass of 40 attograms. Reactions are measured over time periods as long as 3 hours with high stability, showing a scatter corresponding to a fluid refractive index fluctuation of ± 4 × 10(-6) in the baseline data. Sensor arrays with a SU8 protective cladding are easy to fabricate, while oxide cladding is found to provide superior stability for measurements involving long time scales.

Efficient Nonlinear Solvers for Nodal High-Order Finite Elements in 3D

Conventional high-order finite element methods are rarely used for industrial problems because the Jacobian rapidly loses sparsity as the order is increased, leading to unaffordable solve times and memory requirements. This effect typically limits order to at most quadratic, despite the favorable accuracy and stability properties offered by quadratic and higher order discretizations. We present a method in which the action of the Jacobian is applied matrix-free exploiting a tensor product basis on hexahedral elements, while much sparser matrices based on Q 1 sub-elements on the nodes of the high-order basis are assembled for preconditioning. With this “dual-order” scheme, storage is independent of spectral order and a natural taping scheme is available to update a full-accuracy matrix-free Jacobian during residual evaluation. Matrix-free Jacobian application circumvents the memory bandwidth bottleneck typical of sparse matrix operations, providing several times greater floating point performance and better use of multiple cores with shared memory bus. Computational results for the p-Laplacian and Stokes problem, using block preconditioners and AMG, demonstrate mesh-independent convergence rates and weak (bounded) dependence on order, even for highly deformed meshes and nonlinear systems with several orders of magnitude dynamic range in coefficients. For spectral orders around 5, the dual-order scheme requires half the memory and similar time to assembled quadratic (Q 2) elements, making it very affordable for general use.

Leather Structure Determination by Small-Angle X-ray Scattering (SAXS): Cross Sections of Ovine and Bovine Leather

SAXS has been applied to structural determination in leather. The SAXS beamline at the Australian Synchrotron provides 6 orders of magnitude dynamic range, enabling a rich source of structural information from scattering patterns of leather sections. SAXS patterns were recorded for q from 0.004 to 0.223 A(-1). Collagen d spacing varied across ovine leather sections from 63.8 nm in parts of the corium up to 64.6 nm in parts of the grain. The intensity of the collagen peak at q = 0.06 A(-1) varied by 1 order of magnitude across ovine leather sections with the high-intensity region in the corium and the low intensity in the grain. The degree of fiber orientation and the dispersion of the orientation has been quantified in leather. It is shown how the technique provides a wealth of useful information that may be used to characterize and compare leathers, skin, and connective tissue.

Resolving the Composition of Protein Complexes Using a MALDI LTQ Orbitrap

Current biological studies have been advanced by the continuous development of robust, accurate, and sensitive mass spectrometric technologies. The MALDI LTQ Orbitrap is a new addition to the Orbitrap configurations, known for their high resolving power and accuracy. This configuration provides features inherent to the MALDI source, such as reduced spectra complexity, forgiveness to contaminants, and sample retention for follow-up analyses with targeted or hypothesis-driven questions. Here we investigate its performance for characterizing the composition of isolated protein complexes. To facilitate the assessment, we selected two well characterized complexes from Saccharomyces cerevisiae, Apl1 and Nup84. Manual and automatic MS and MS/MS analyses readily resolved their compositions, with increased confidence of protein identification compared with our previous reports using MALDI QqTOF and MALDI IT. CID fragmentation of singly-charged peptides provided sufficient information for conclusive identification of the isolated proteins. We then assessed the resolution, accuracy, and sensitivity provided by this instrument in the context of analyzing the isolated protein assemblies. Our analysis of complex mixtures of singly-charged ions up to m/z 4000 showed that (1) the resolving power, inversely proportional to the square root of m/z, had over four orders of magnitude dynamic range; (2) internal calibration led to improved accuracy, with an average absolute mass error of 0.5 ppm and a distribution centered at 0 ppm; and (3) subfemtomole sensitivity was achieved using both CHCA and DHB matrices. Additionally, our analyses of a synthetic phosphorylated peptide in mixtures showed subfemtomole level of detection using neutral loss scanning.

High-speed charge-to-time converter ASIC for the Super-Kamiokande detector

A new application-specific integrated circuit (ASIC), the high-speed charge-to-time converter (QTC) IWATSU CLC101, provides three channels, each consisting of preamplifier, discriminator, low-pass filter, and charge integration circuitry, optimized for the waveform of a photomultiplier tube (PMT). This ASIC detects PMT signals using individual built-in discriminators and drives output timing signals whose width represents the integrated charge of the PMT signal. Combined with external input circuits composed of passive elements, the QTC provides full analog signal processing for the detector's PMTs, ready for further processing by time-to-digital converters (TDCs). High-rate ( > 1 MHz ) signal processing is achieved by short-charge-conversion-time and baseline-restoration circuits. Wide-range charge measurements are enabled by offering three gain ranges while maintaining a short cycle time. QTC chip test results show good analog performance, with efficient detection for a single photoelectron signal, four orders of magnitude dynamic range ( 0.3 mV ∼ 3 V ; 0.2 ∼ 2500 pC ), 1% charge linearity, 0.2 pC charge resolution, and 0.1 ns timing resolution. Test results on ambient temperature dependence, channel isolation, and rate dependence also meet specifications.

Six orders of magnitude dynamic range in capillary electrophoresis with ultrasensitive laser-induced fluorescence detection

An ultrasensitive laser-induced fluorescence detector was used with capillary electrophoresis for the study of 5-carboxy-tetramethylrhodamine. The raw signal from the detector provided roughly three orders of magnitude dynamic range. The signal saturated at high analyte concentrations due to the dead time associated with the single-photon counting avalanche photodiode employed in the detector. The signal can be corrected for the detector dead time, providing an additional order of magnitude dynamic range. To further increase dynamic range, two fiber-optic beam-splitters were cascaded to generate a primary signal and two attenuated signals, each monitored by a single-photon counting avalanche photodiode. The combined signals from the three photodiodes are reasonably linear from the concentration detection limit of 3 pM to 10 μM, the maximum concentration investigated, a range of 3,000,000. Mass detection limits were 150 yoctomoles injected onto the capillary.

In situ Pb-isotope analysis of pyrite by laser ablation (multi-collector and quadrupole) ICPMS

Pb-isotope ratios, measured in the mineral pyrite, provide a valuable petrogenetic tool with widespread applicability. In order to interpret complex structural and mineralogical textures, however, a method of in-situ analysis is essential. While laser ablation ICPMS is ideally suited to this task, the low melting point of sulfide, the highly variable and often high Pb contents, and the potential presence of relatively radiogenic inclusions introduce a number of analytical problems unique to pyrite Pb-isotope analysis. Here we address these issues using results obtained on two very different analytical systems based around multi-collector and quadrupole ICPMS instruments respectively. We suggest that controlled ablation of pyrite is only achieved at low laser fluence and that, under these conditions, standardisation using silicate reference materials is inappropriate and natural pyrite standards are to be preferred. The inherent variability in Pb (and sometimes U) concentrations in pyrite requires careful selection of detector systems for optimal analysis and in this regard both quadrupole and multi-collector ICPMS instruments can play important and complimentary roles. Multi-collector instruments provide higher precision analyses but detector configurations can prohibit simultaneous measurement of U, Th and Pb. Furthermore, the micrometer-scale variability in Pb concentrations can cause problems for both Faraday cup and ion counting detection systems. In contrast, quadrupole ICPMS systems allow simultaneous measurement of U, Th and Pb, and have more flexible detection systems with many orders of magnitude dynamic range but are unable to produce high precision data. Results are presented for two different analytical systems and demonstrate a very strong dependence of data quality upon signal size. In addition they allow some estimation of the limiting precision obtainable by these methods. Finally, a geological example is provided from the giant Sukhoi Log sedimentary Au deposit of Russia.

Dynamically multiplexed ion mobility time-of-flight mass spectrometry.

Ion mobility spectrometry-time-of-flight mass spectrometry (IMS-TOFMS) has been increasingly used in analysis of complex biological samples. A major challenge is to transform IMS-TOFMS to a high-sensitivity, high-throughput platform, for example, for proteomics applications. In this work, we have developed and integrated three advanced technologies, including efficient ion accumulation in an ion funnel trap prior to IMS separation, multiplexing (MP) of ion packet introduction into the IMS drift tube, and signal detection with an analog-to-digital converter, into the IMS-TOFMS system for the high-throughput analysis of highly complex proteolytic digests of, for example, blood plasma. To better address variable sample complexity, we have developed and rigorously evaluated a novel dynamic MP approach that ensures correlation of the analyzer performance with an ion source function and provides the improved dynamic range and sensitivity throughout the experiment. The MP IMS-TOFMS instrument has been shown to reliably detect peptides at a concentration of 1 nM in the presence of a highly complex matrix, as well as to provide a 3 orders of magnitude dynamic range and a mass measurement accuracy of better than 5 ppm. When matched against human blood plasma database, the detected IMS-TOF features were found to yield approximately 700 unique peptide identifications at a false discovery rate (FDR) of approximately 7.5%. Accounting for IMS information gave rise to a projected FDR of approximately 4%. Signal reproducibility was found to be greater than 80%, while the variations in the number of unique peptide identifications were <15%. A single sample analysis was completed in 15 min that constitutes almost 1 order of magnitude improvement compared to a more conventional LC-MS approach.

A user-friendly, highly sensitive assay to detect the IFN-γ secretion by T cells

Objectives Development of a user-friendly test alternative to ELISA-based assays to detect IFN-γ by in vitro cultured peripheral blood mononuclear cells (PBMC) stimulated with pathogen-derived antigens. Design and methods The molecular components of an operational IFN-γ ELISA-based test were applied in a lateral flow (LF) immuno-sandwich assay using up-converting phosphor (UCP) reporter particles. The analytical sensitivity of the UCP-LF IFN-γ assay (ULIGA) was determined and the assay was qualitatively validated with a selection of 60 supernatants derived from PBMC cultures stimulated with M. leprae derived antigens, mitogen or medium alone. Results ULIGA indicated an analytical sensitivity better than 2 pg/mL, and demonstrated four orders of magnitude dynamic range. The assay correlated well with the IFN-γ ELISA. Conclusions ULIGA allows detection well below the cutoff value (100 pg/mL) used to define positive responses in the IFN-γ ELISA. The test procedure is less demanding in respect to equipment and labor, and is suited for testing single samples.

NONLINEAR TRANSMISSION OF PHOTOSENSITIVE CHOLESTERIC LIQUID CRYSTALS DUE TO SPECTRAL BANDWIDTH AUTO-TUNING OR RESTORATION

Cholesteric liquid crystals (CLCs) which possess a periodicity in the visible portion of the spectra, exhibit selective reflection of circularly polarized light. The ability to modulate this color through a variety of means has been explored, including work which incorporated azobenzene LCs. Two types of systems have recently been explored utilizing wavelength-specific cis-trans isomerization processes, which enable unprecedented photosensitivity. The first system exhibits large blue or red-shifted changes in reflection wavelength upon visible irradiation. The second system exploits the metastable, long-lived photoinduced isotropic state, whose return to the reflective Grandjean texture can be induced by wavelength-specific radiation. We demonstrate nonlinear transmission from both types of systems, starting with submicrowatt power levels and spanning over four orders of magnitude dynamic range. The power dependence and temporal evolution of this effect (10–100 ms) is documented here for red or green laser wavelengths. The effect for the former case is due to bandgap auto-tuning, when the laser beam is tuning the CLC Bragg reflection band to its own wavelength. For the latter case, autonomous, optical feedback due to bandgap restoration is the cause of the nonlinear transmission properties.

Enhanced sequence coverage of proteins in human cerebrospinal fluid using multiple enzymatic digestion and linear ion trap LC-MS/MS

The cerebrospinal fluid (CSF) provides a ready access into the health state of the central nervous system, and alterations in some CSF proteins have been documented in brain disease. However, the complete variety of proteins is not known and methods to identify protein components are still being developed. The goal of this study was to examine the sequence coverage obtained from human CSF digests produced with different proteases. Enzymatic digests of CSF proteins were obtained with arginine-C endopeptidase (ArgC), glutamic acid endopeptidase (GluC), chymotrypsin, trypsin and their combinations, and then examined using reverse phase chromatography and a Finnigan LTQ linear ion trap mass spectrometer. Peptide sequences were identified with BioWorks 3.1 and sequence coverage calculated for the 38 most confidently identified proteins. Trypsin and GluC yielded greater coverage than chymotrypsin, while ArgC had the least sequence coverage. Protein sequence coverage was affected only slightly over four orders of magnitude dynamic range of abundance. Combining the peptides derived from different proteases further increased the coverage. Maximal sequence coverage was achieved by combining digest results from both GluC and trypsin. These results have implications for future studies to identify CSF proteins and their post-translational modifications.