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In complex mixtures, proton nuclear magnetic resonance (NMR) spectra are often very crowded, making spectral analysis complicated or even impossible, particularly when detailed structural information about the mixture components is needed. A new 1D NMR method (fluorine-edited selective TOCSY acquisition, FESTA) is introduced that facilitates the structural analysis of mixtures of species that contain fluorine. It allows simplified 1H spectra to be obtained that show only those protons that are in a spin system coupled to fluorine of interest. The new method is illustrated by factorizing a complex ¹H spectrum into subspectra for individual spin systems involving different 19F sites. [ABSTRACT FROM AUTHOR]

A method for multiplexed sample analysis by mass spectrometry without the need for chemical tagging is presented. In this new method, each sample is pulsed at unique frequencies, mixed, and delivered to the mass spectrometer while maintaining a constant total flow rate. Reconstructed ion currents are then a time-dependent signal consisting of the sum of the ion currents from the various samples. Spectral deconvolution of each reconstructed ion current reveals the identity of each sample, encoded by its unique frequency, and its concentration encoded by the peak height in the frequency domain. This technique is different from other approaches that have been described, which have used modulation techniques to increase the signal-to-noise ratio of a single sample. As proof of concept of this new method, two samples containing up to 9 analytes were multiplexed. The linear dynamic range of the calibration curve was increased with extended acquisition times of the experiment d longer oscillation periods of the samples. Because of the combination of the samples, salt had little effect on the ability of this method to achieve relative quantitation. Continued development of this method is expected to allow for increased numbers of samples that can be multiplexed. [ABSTRACT FROM AUTHOR]

The interface of nucleic acids and nanomaterials is among the most promising fields in recent years. Considerable efforts have been devoted to the development of novel systems based on the two components for various promising applications such as sensing, bioimaging, drug delivery, and theranostics. However, the determination of nucleic acid concentration in these systems remains as a challenge due to the interference of nanoparticles. To this end, we developed a simple, yet reliable, method to quantify the nucleic acid concentration in their nanoparticle or polymer conjugates based on circular dichroism (CD) spectroscopy. In this paper, three nucleic acids, namely, DNA sodium salt from calf thymus (NaDNA), DNA from herring sperm (hsDNA), and ribonucleic acid from torula yeast (tyRNA), were noncovalently conjugated to three nanoparticles. The concentrations of the three nucleic acids in their nanoparticle conjugates were successfully determined on the basis of CD spectra calibration curves. [ABSTRACT FROM AUTHOR]

A single platform, integrated by a laser-induced breakdown spectroscopy detector and a Raman spectroscopy sensor, has been designed to remotely (5 m) and simultaneously register the elemental and molecular signatures of rocks under Martian surface conditions. From this information, new data fusion architecture at decisions level is proposed for the correct categorization of the rocks. The approach is based on a decision-making process from the sequential checking of the spectral features representing the cationic and anionic counterparts of the specimen. The scrutiny of the LIBS response by using a moving-window algorithm informs on the diversity of the elemental constituents. The output rate of emission lines allows projecting in a loop the elements as the cationic counterpart of the rock. In parallel, the Raman response of the unknown is compared with all the molecular counterparts of the hypothesized cation that are stored in a spectral library. The largest similarity rate unveils the final identity of the unknown. The identification capabilities of the architecture have been underscored through blind tests of 10 natural rocks with different origins. The great majority of forecasts have matched with the real identities of the inspected targets. The strength of this platform to simultaneously acquire the multielemental and the molecular information from a specimen by using the same laser events greatly enhances the "on-surface" missions for the surveillance of mineralogy. [ABSTRACT FROM AUTHOR]

In this work, we demonstrate a novel design that allows rapid online identification and quantitation of unsaturated fatty acid C=C location isomers via epoxidation and ambient mass spectrometry (MS). Unsaturated fatty acid solution was loaded on a paper strip placed between a low-temperature plasma probe and the inlet of a mass spectrometer. Reactive oxygen species in the plasma promoted epoxidation at the C=C, and the product was simultaneously ionized. Upon collision-induced dissociation (CID), the epoxidation product was fragmented to release diagnostic ions specific to the C=C location. The whole analytical workflow can be completed within 5 s and is particularly promising for point-of-care (POC) clinical diagnosis, considering its fast, high-throughput nature, and coupling with paper-based analytical devices. [ABSTRACT FROM AUTHOR]

Following a brief overview of the emergence of absorbance detection in liquid chromatography, we focus on the dispersion caused by the absorbance measurement cell and its inlet. A simple experiment is proposed wherein chromatographic flow and conditions are held constant but a variable portion of the column effluent is directed into the detector. The temporal peak variance (σ2t,obs), which increases as the flow rate (F) through the detector decreases, is found to be well-described as a quadratic function of 1/F. This allows the extrapolation of the results to zero residence time in the detector and thence the determination of the true variance of the peak prior to the detector (this includes contribution of all preceding components). This general approach should be equally applicable to detection systems other than absorbance. We also experiment where the inlet/outlet system remains the same but the path length is varied. This allows one to assess the individual contributions of the cell itself and the inlet/outlet system.to the total observed peak. The dispersion in the cell itself has often been modeled as a flow-independent parameter, dependent only on the cell volume. Except for very long path/large volume cells, this paradigm is simply incorrect. [ABSTRACT FROM AUTHOR]

Real-time in situ mass spectrometry analysis of airborne particles is important in several applications, including exposure studies in ambient air, industrial settings, and assessing impacts on visibility and climate. However, obtaining molecular and 3D structural information is more challenging, especially for heterogeneous solid or semisolid particles. We report a study of extractive electrospray ionization mass spectrometry (EESI-MS) for the analysis of solid particles with an organic coating. The goal is to elucidate how much of the overall particle content is sampled, and determine the sensitivity of this technique to the surface layers. It is shown that, for NaNO3 particles coated with glutaric acid (GA), very little of the solid NaNO3 core is sampled compared to the GA coating, whereas for GA particles coated with malonic acid (MA), significant signals from both the MA coating and the GA core are observed. However, conventional ESI-MS of the same samples collected on a Teflon filter (and then extracted) detects much more core material compared to EESI-MS in both cases. These results show that, for the experimental conditions used here, EESI-MS does not sample the entire particle but, instead, is more sensitive to surface layers. Separate experiments on single-component particles of NaNO3, GA, or citric acid show that there must be a kinetics limitation to dissolution that is important in determining EESI-MS sensitivity. We propose a new mechanism of EESI solvent droplet interaction with solid particles that is consistent with the experimental observations. In conjunction with previous EESI-MS studies of organic particles, these results suggest that EESI does not necessarily sample the entire particle when solid, and that not only solubility but also surface energies and the kinetics of dissolution play an important role. [ABSTRACT FROM AUTHOR]

We describe complex organic mixture analysis by 21 tesla (T) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ultrahigh mass-resolving power (m/Δm50% > 2 700 000 at m/z 400) and mass accuracy (80 ppb rms) enable resolution and confident identification of tens of thousands of unique elemental compositions. We demonstrate 2.2-fold higher mass-resolving power, 2.6-fold better mass measurement accuracy, and 1.3-fold more assigned molecular formulas compared to our custom-built, state-of-theart 9.4 T FT-ICR mass spectrometer for petroleum and dissolved organic matter (DOM) analyses. Analysis of a heavy petroleum distillate exemplifies the need for ultrahigh-performance mass spectrometry (49 040 assigned molecular formulas for 21 T versus 29 012 for 9.4 T) and extends the identification of previously unresolved Oo, SsOo, and NOo classes. Mass selective ion accumulation (20 Thompson isolation) of an asphalt volcano sample yields 462 resolved mass spectral peaks at m/z 677 and reveals previously unresolved CcHhNnOoSs mass differences at high mass (m/z > 600). Similar performance gains are realized in the analysis of dissolved organic matter, where doubly charged Oo species are resolved from singly charged SOo species, which requires a mass-resolving power greater than 1 400 000 (at m/z 600). This direct comparison reveals the continued need for higher mass-resolving power and better mass accuracy for comprehensive molecular characterization of the most complex organic mixtures. [ABSTRACT FROM AUTHOR]

We present a high-throughput screening Raman spectroscopy (HTS-RS) platform for a rapid and label-free macromolecular fingerprinting of tens of thousands eukaryotic cells. The newly proposed label-free HTS-RS platform combines automated imaging microscopy with Raman spectroscopy to enable a rapid label-free screening of cells and can be applied to a large number of biomedical and clinical applications. The potential of the new approach is illustrated by two applications. (1) HTS-RS-based differential white blood cell count. A classification model was trained using Raman spectra of 52 218 lymphocytes, 48 220 neutrophils, and 7 294 monocytes from four volunteers. The model was applied to determine a WBC differential for two volunteers and three patients, producing comparable results between HTS-RS and machine counting. (2) HTS-RS-based identification of circulating tumor cells (CTCs) in 1:1, 1:9, and 1:99 mixtures of Panc1 cells and leukocytes yielded ratios of 55:45, 10:90, and 3:97, respectively. Because the newly developed HTS-RS platform can be transferred to many existing Raman devices in all laboratories, the proposed implementation will lead to a significant expansion of Raman spectroscopy as a standard tool in biomedical cell research and clinical diagnostics. [ABSTRACT FROM AUTHOR]

The increasing focus on lipid metabolism has revealed a need for analytical techniques capable of structurally characterizing lipids with a high degree of specificity. Lipids can exist as any one of a large number of double bond positional isomers, which are indistinguishable by single-stage mass spectrometry alone. Ozonolysis reactions coupled to mass spectrometry have previously been demonstrated as a means for localizing double bonds in unsaturated lipids. Here we describe an online, solution-phase reactor using ozone produced via a low-pressure mercury lamp, which generates aldehyde products diagnostic of cleavage at a particular double bond position. This flow-cell device is utilized in conjunction with structurally selective ion mobility-mass spectrometry. The lamp-mediated reaction was found to be effective for multiple lipid species in both positive and negative ionization modes, and the conversion efficiency from precursor to product ions was tunable across a wide range (20-95%) by varying the flow rate through the ozonolysis device. Ion mobility separation of the ozonolysis products generated additional structural information and revealed the presence of saturated species in a complex mixture. The method presented here is simple, robust, and readily coupled to existing instrument platforms with minimal modifications necessary. For these reasons, application to standard lipidomic workflows is possible and aids in more comprehensive structural characterization of a myriad of lipid species. [ABSTRACT FROM AUTHOR]

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source coupled to the Q Exactive Plus has been extensively used in untargeted mass spectrometry imaging (MSI) analyses of biological tissue sections. Although the Orbitrap is a high-resolution and accurate-mass (HRAM) mass analyzer, these attributes alone cannot be used for the reliable identification of unknown analytes observed in complex biological matrices. Spectral accuracy (SA) is the ability of the mass spectrometer to accurately measure the isotopic distributions which, when used with high mass measurement accuracy (MMA), can facilitate the elucidation of a single elemental composition. To investigate the effects of different ion populations on an Orbitrap's SA and MMA, a solution of caffeine, the tetrapeptide MRFA, and ultramark was analyzed using a Q Exactive Plus across eight distinct automatic gain control (AGC) targets. The same compounds from the same lot numbers were also individually analyzed using isotope ratio mass spectrometry (IRMS) to accurately determine the isotopic abundance of 13C, 15N, and 34S. We demonstrated that at optimum absolute ion abundances the Orbitrap can be used to accurately count carbons, nitrogens, and sulfurs in samples with varying masses. Additionally, absolute monoisotopic ion abundances required for high SA were empirically determined by using the expected (IRMS) and experimental (Orbitrap) isotopic distributions to calculate the Pearson chi-square test. These thresholds for absolute ion abundances can be used in untargeted MSI studies to shorten an identification list by rapidly screening for isotopic distributions whose absolute ion abundances are high enough to accurately estimate the number of atoms. [ABSTRACT FROM AUTHOR]

Intracellular pH (pHi) is an important parameter associated with cellular behaviors and pathological conditions. Sensing pHi and monitoring its changes are essential but challenging due to the lack of high-sensitive probes. Herein, a ratiometric fluorescent probe with ultra pH-sensitivity is developed based on hairpin-contained i-motif strand (I-strand, labeled with Rhodamine Green and BHQ at two termini) and complementary strand (C-strand, labeled with Rhodamine Red at its 5'-end). At neutral pH, both I-strand and C-strand hybridize into a rigid duplex (I-C), which holds the Rhodamine Red and the BHQ2 in close proximity. As a result, the fluorescence emission (F597 nm) of the Rhodamine Red is strongly suppressed, while the Rhodamine Green (F542 nm) is in a "signal on" state. However, the slightly acidic pH enforced the I-strand to form an intramolecular i-motif and initiated the dehybridization of I -- C duplex, leading to Rhodamine Red in a "signal on" state and a decreased fluorescence of Rhodamine Green. The ratio (F542 nm/F597 nm) can be used as a signal for pH sensing. Due to the rational internal hairpin design of I -- C duplex probe, almost 70-fold change in the ratio was observed in the physiological pH range (6.50-7.40). This probe possesses efficient stability, fast response, and reversible pH measurement capabilities. Furthermore, intracellular application of the ratiometric probe was demonstrated on the example of SMMC-7721 cells. With different recognition elements in engineering of i-motif based platforms, the design might hold great potential to become a versatile strategy for intracellular pH sensing. [ABSTRACT FROM AUTHOR]

Stable-isotope labeling experiments (ILEs) are widely used to investigate the topology and operation of metabolic networks. The quality of isotopic data collected in ILEs is of utmost importance to ensure reliable biological interpretations, but current evaluation approaches are limited due to a lack of suitable reference material and relevant evaluation criteria. In this work, we present a complete methodology to evaluate mass spectrometry (MS) methods used for quantitative isotopic studies of metabolic systems. This methodology, based on a biological sample containing metabolites with controlled labeling patterns, exploits different quality metrics specific to isotopic analyses (accuracy and precision of isotopologue masses, abundances, and mass shifts and isotopic working range). We applied this methodology to evaluate a novel LC-MS method for the analysis of amino acids, which was tested on high resolution (Orbitrap operating in full scan mode) and low resolution (triple quadrupole operating in multiple reaction monitoring mode) mass spectrometers. Results show excellent accuracy and precision over a large working range and revealed matrix-specific as well as mode-specific characteristics. The proposed methodology can identify reliable (and unreliable) isotopic data in an easy and straightforward way and efficiently supports the identification of sources of systematic biases as well as of the main factors that influence the overall accuracy and precision of measurements. This approach is generic and can be used to validate isotopic analyses on different matrices, analytical platforms, labeled elements, or classes of metabolites. It is expected to strengthen the reliability of isotopic measurements and thereby the biological value of ILEs. [ABSTRACT FROM AUTHOR]

Quantitative detection of impurities in organic crystals was demonstrated by accurately measuring absorption frequencies using a continuous wave gallium phosphide terahertz spectrometer. THz spectra of L-asparagine monohydrate doped with L-aspartic acid at 0.05 to 12.5 wt % were obtained at 10 IK The three lowest frequency absorption peaks were baseline-resolved, allowing them to be examined independently. Using a least-squares curve fitting technique, impurities were detected at levels as low as 500 ppm. The sensitivity and detection limits of the technique depended strongly on the nature of both the host and the impurities. The projected limit of detection using the current system, given optimal materials, was estimated to be 51.7 ppm. In addition to quantitative assessments, impurities may also be identified by comparing frequency shifts of multiple absorptions. [ABSTRACT FROM AUTHOR]

Two-dimensional (2D) J-resolved NMR technique offers a natural solution for disentangling complex mixtures that suffer from crowded spectra in 1D NMR. The applicability of classical 2D J-resolved spectroscopy is inevitably limited by phase-twist lineshapes and strong coupling artifacts. Here, a general and robust NMR method is proposed to record 2D absorption-mode J-resolved spectra in rapid acquisition manner. This method can also reduce the impact of strong coupling artifacts, thus achieving full considerations for applications. Intuitively, this method delivers pure chemical shifts along one dimension and orthogonally adds J couplings along the other dimension, free of 45° spectral shearing. It may provide a powerful tool for structural and configurational studies as well as biological analyses. [ABSTRACT FROM AUTHOR]

The use of collision cross-section (CCS) values derived from ion mobility-mass spectrometry (IM-MS) has been proven to facilitate lipid identifications. Its utility is restricted by the limited availability of CCS values. Recently, the machine-learning algorithm-based prediction (e.g., MetCCS) is reported to generate CCS values in a large-scale. However, the prediction precision is not sufficient to differentiate lipids due to their high structural similarities and subtle differences on CCS values. To address this challenge, we developed a new approach, namely, LipidCCS, to precisely predict lipid CCS values. In LipidCCS, a set of molecular descriptors were optimized using bioinformatic approaches to comprehensively describe the subtle structure differences for lipids. The use of optimized molecular descriptors together with a large set of standard CCS values for lipids (458 in total) to build the prediction model significantly improved the precision. The prediction precision of LipidCCS was externally validated with median relative errors (MRE) of ~1% using independent data sets across different instruments (Agilent DTIM-MS and Waters TWIM-MS) and laboratories. We also demonstrated that the improved precision in the predicted LipidCCS database (15?646 lipids and 63?434 CCS values in total) could effectively reduce false-positive identifications of lipids. Common users can freely access our LipidCCS web server for the following: (1) the prediction of lipid CCS values directly from SMILES structure; (2) database search; and (3) lipid match and identification. We believe LipidCCS will be a valuable tool to support IM-MS-based lipidomics. The web server is freely available on the Internet (http://www.metabolomics-shanghai.org/LipidCCS/). [ABSTRACT FROM AUTHOR]

Collision cross section (CCS) measurements resulting from ion mobility-mass spectrometry (IM-MS) experiments provide a promising orthogonal dimension of structural information in MS-based analytical separations. As with any molecular identifier, interlaboratory standardization must precede broad range integration into analytical workflows. In this study, we present a reference drift tube ion mobility mass spectrometer (DTIM-MS) where improvements on the measurement accuracy of experimental parameters influencing IM separations provide standardized drift tube, nitrogen CCS values ([sup DT]CCS[sub N2]) for over 120 unique ion species with the lowest measurement uncertainty to date. The reproducibility of these [sup DT]CCS[sub N2] values are evaluated across three additional laboratories on a commercially available DTIM-MS instrument. The traditional stepped field CCS method performs with a relative standard deviation (RSD) of 0.29% for all ion species across the three additional laboratories. The calibrated single field CCS method, which is compatible with a wide range of chromatographic inlet systems, performs with an average, absolute bias of 0.54% to the standardized stepped field [sup DT]CCS[sub N2] values on the reference system. The low RSD and biases observed in this interlaboratory study illustrate the potential of DTIM-MS for providing a molecular identifier for a broad range of discovery based analyses. [ABSTRACT FROM AUTHOR]

Ion mobility-mass spectrometry (IM-MS) can provide orthogonal information, i.e., m/z and collision cross section (CCS), for the identification of drugs and drug metabolites. However, only a small number of CCS values are available for drugs, which limits the use of CCS as an identification parameter and the assessment of structure-function relationships of drugs using IM-MS. Here, we report the development of a rapid workflow for the measurement of CCS values of a large number of drug or drug-like molecules in nitrogen on the widely available traveling wave IM-MS (TWIM-MS) platform. Using a combination of small molecule and polypeptide CCS calibrants, we successfully determined the nitrogen CCS values of 1425 drug or drug-like molecules in the MicroSource Discovery Systems' Spectrum Collection using flow injection analysis of 384-well plates. Software was developed to streamline data extraction, processing, and calibration. We found that the overall drug collection covers a wide CCS range for the same mass, suggesting a large structural diversity of these drugs. However, individual drug classes appear to occupy a narrow and unique space in the CCS-mass 2D spectrum, suggesting a tight structure-function relationship for each class of drugs with a specific target. We observed bimodal distributions for several antibiotic species due to multiple protomers, including the known fluoroquinolone protomers and the new finding of cephalosporin protomers. Lastly, we demonstrated the utility of the high-throughput method and drug CCS database by quickly and confidently confirming the active component in a pharmaceutical product. [ABSTRACT FROM AUTHOR]

Femtosecond stimulated Raman spectroscopy (FSRS) is a vibrational spectroscopy technique that has been used in a wide variety of applications: from transient vibrational signature tracking to amplifying weak normal Raman scattering signals. Presented here is an application of FSRS to quantify the differential Raman scattering cross sections (DRSCs) of glucose. In using FSRS to determine the DRSCs of multiple glucose vibrational modes, we demonstrate the applicability of both stimulated Raman loss (SRL) spectroscopy and stimulated Raman gain (SRG) FSRS. Using the two analogous FSRS techniques, SRG and SRL, we determine that the DRSCs of glucose excited at 514.5 nm range from a low of 5.0 ± 1.1 × 10-30 to a high of 8.9 ± 0.9 × 10-30 cm2 molecule-1 sr-1. This work establishes both the compatibility of SRL for measuring DRSCs and values for the DRSC of multiple vibrational modes of glucose. [ABSTRACT FROM AUTHOR]

Electrochemical deposition of aromatic organic molecules by reduction of diazonium reagents enables formation of molecular layers with sufficient integrity for use in molecular electronic junctions of interest to microelectronics. Characterization of organic films with thicknesses in the 1-10 nm range is difficult with Raman spectroscopy, since most molecular structures of electronic interest have Raman cross sections which are too small to observe as either thin films on solid electrodes or within intact molecular junctions. Layer formation on a 10 nm thick Ag island film on a flat carbon surface (eC/Ag) permitted acquisition of structural information using surface enhanced Raman spectroscopy (SERS), in many cases for molecules with weak Raman scattering. Raman spectra obtained on eC/Ag surfaces were indistinguishable from those on carbon without Ag present, and the spectra of oligomeric molecular layers were completely consistent with those of the monomers. Layer growth was predominantly linear for cases where such growth was sterically allowed, and linear growth correlated strongly with the line width and splitting of the C?C phenyl ring stretches. Molecular bilayers made by successive reduction of different diazonium reagents were also observable and will be valuable for applications of 1-20 nm organic films in molecular electronics. [ABSTRACT FROM AUTHOR]

Polarization-resolved Raman spectroscopy provides much more information than its conventional counterpart. However, it usually either requires a complicated setup with two spectrographs and detectors or two measurements must be performed sequentially. This study presents a simple and straightforward approach to recording both polarization components simultaneously with a single spectrograph and detector. The vertically and a horizontally polarized laser beam exiting a Wollaston prism are focused into the sample with a small spatial separation. The scattered light from both beams is imaged onto the slit of an imaging spectrograph as two spatially separated signals, i.e., the polarized and the depolarized Raman signal. Eventually, both spectra are acquired on a single CCD chip simultaneously. Experimental data of ethanol and dimethyl sulfoxide are shown as proof-of-concept. The new method has a number of advantages, for example, laser intensity fluctuations and the polarization dependence of the diffraction grating do not play a role. The proposed approach will be useful for an improved structural analysis and it will be the enabling technology for temporally resolved enantioselective Raman (esR) spectroscopy. [ABSTRACT FROM AUTHOR]

Orbitraps are high-resolution ion-trap mass spectrometers that are widely used in metabolomics. While the mass accuracy and resolving power of orbitraps have been extensively documented, their spectral accuracy, i.e., accuracy in measuring the abundances of isotopic peaks, remains less studied. In analyzing spectra of unlabeled metabolites, we discovered a systematic under representation of heavier natural isotopic species, especially for high molecular weight metabolites (~20% under-measurement of [M + 1]/[M + 0] ratio at m/z 600). We hypothesize that these discrepancies arise for metabolites far from the lower limit of the mass scan range, due to the weaker containment in the C-trap that results in suboptimal trajectories inside the Orbitrap analyzer. Consistent with this, spectral fidelity was restored by dividing the mass scan range (initially 75 m/z to 1000 m/z) into two scan events, one for lower molecular weight and the other for higher molecular weight metabolites. Having thus obtained accurate mass spectra at high resolution, we found that natural isotope correction for high-resolution labeling data requires more sophisticated algorithms than typically employed: the correction algorithm must take into account whether isotopologues with the same nominal mass are resolved. We present an algorithm and associated open-source code, named AccuCor, for this purpose. Together, these improvements in instrument parameters and natural isotope correction enable more accurate measurement of metabolite labeling and thus metabolic flux. [ABSTRACT FROM AUTHOR]

Assessment of airway secretion cells, both for research and clinical purposes, is a highly desired goal in patients with acute and chronic pulmonary diseases. However, lack of proper cell isolation and enrichment techniques hinder downstream evaluation and characterization of cells found in airway secretions. Here, we demonstrate a novel enrichment method to capture immune-related cells from clinical airway secretions using closed-loop separation of spiral inertial microfluidics (C-sep). By recirculating the output focusing stream back to the input reservoir and running continuously with a high flow processing rate, one can achieve optimal concentration, recovery and purity of airway immune cells from a large volume of diluent, which was not readily possible in the single-pass operation. Our method reproducibly recovers 94.0% of polymorphonuclear leukocytes (PMNs), with up to 105 PMNs in clear diluted buffer from 50 μL of airway secretions obtained from mechanically ventilated patients. We show that C-sep isolated PMNs show higher neutrophil elastase (NE) release following activation by phorbol 12-myristate 13-acetate (PMA) than cells isolated by conventional mucolytic method. By capturing cells without chemically disrupting their potential function, our method is expected to expand the possibility of clinical in vitro cell based biological assays for various pulmonary diseases such as acute respiratory distress syndrome, pneumonia, cystic fibrosis, and bronchiectasis. [ABSTRACT FROM AUTHOR]

Phthalic acid esters (PAEs) are ubiquitous in the environment, and some of them are recognized as endocrine disruptors that cause concerns on ecosystem functioning and public health. Due to the diversity of PAEs in the environment, there is a vital need to detect the total concentration of PAEs in a timely and low-cost way. To fulfill this requirement, it is highly desired to obtain group-specific PAE binders that are specific to the basic PAE skeleton. In this study, for the first time we have identified the group-specific PAE-binding aptamers via rationally designed target immobilization. The two target immobilization strategies were adopted to display either the phthalic ester group or the alkyl chain, respectively, at the surface of the immobilization matrix. The former enabled the rapid enrichment of aptamers after four rounds of selection. The top 100 sequences are cytosine-rich (44.7%) and differentiate from each other by only 1-4 nucleotides at limited locations. The top two aptamers all display the nanomolar dissociation constants to both the immobilized target and the free PAEs [dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), bis(2-ethylhexyl) phthalate (DEHP)]. We further demonstrate the applications of the aptamers in the development of high-throughput PAE assays and DEHP electrochemical biosensors with exceptional sensitivity [limit of detection (LOD), 10 pM] and selectivity (>105-fold). PAE aptamers targeting one of the most sought for targets thus offer the promise of convenient, low-cost detection of total PAEs. Our study also provides insights on the aptamer selection and sensor development of highly hydrophobic small molecules. [ABSTRACT FROM AUTHOR]

Two-dimensional infrared (2D IR) spectroscopy provides a powerful approach for the direct study of molecular dynamics with high spatial and temporal resolution. Its application for investigating specific locations in proteins requires the incorporation of IR probe groups with spectrally isolated absorptions to avoid the congestion inherent to protein spectra. This has motivated extensive efforts toward the development of new IR probes, but there remains a need for those that can extend the experimental time range, which is limited by their vibrational lifetimes. Toward this goal, isotopically labeled p-(13C15N-cyano)phenylalanine was synthesized, site-selectively incorporated into the protein plastocyanin, and evaluated for its potential as a 2D IR probe. The isotopic labeling increases the vibrational lifetime about 2-fold, which results in larger signals at longer time scales. However, isotopic labeling simultaneously shifts the absorption to a spectral region with greater water absorbance, which results in greater heating-induced signals in the background that overlap those of the nitrile probe. The study demonstrates the use of a new 2D IR probe to measure the side chain dynamics in a protein and also illustrates the multiple factors to consider in development of 2D IR probes for studying proteins. [ABSTRACT FROM AUTHOR]

Bipolar electrochemistry is receiving growing attention in the last years, not only because it is an important tool for studying electron transfer processes, but also because it is really fruitful in the development of new analytical sensors. Bipolar electrodes show promising applications as a direct analytical tool since oxidation and reduction reactions take place simultaneously on different parts of a single conductor. There are several electrochemical devices that provide information about electron transfer between two immiscible electrolyte solutions, but to the best of our knowledge, this is the first time that a bipolar device is able to record two spectroelectrochemical responses concomitantly at two different compartments. It allows deconvolving the electrochemical signal into two different optical signals related to the electron transfer processes occurring at two compartments that are electrically in contact. The combination of an electrochemical and two spectroscopic responses is indeed very useful, providing essential advantages in the study of a huge variety of systems. The study of three different electrochemical systems, such as reversible redox couples, carbon nanotubes, and conducting polymers has allowed us to validate the new cell and to demonstrate the capabilities of this technique to obtain valuable time-resolved information related to the electron transfer processes. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play crucial roles in the regulation of cellular activities and are next-generation biomarkers for early cancer detection. Simultaneous monitoring of multiplexed miRNA is very important for enhancing the accuracy of cancer diagnostics. Traditional fluorescence methods for multicomponent analysis were usually operated under multiple excitation wavelengths, because spectral crosstalk is very detrimental to detecting accuracy for multicomponent analysis. Herein, we present a fluorescence strategy for multi-miRNAs detection in plasma under a single excitation wavelength. Nucleic acid stain TOTO-1 and three labeled fluorescence dyes Cy3, Cy3.5, and Cy5 emit no fluorescence in their free state. Target miRNA hybridized the auxiliary and probe oligonucleotides into duplex nucleic acid. Intercalation interaction localized TOTO-1 and labeled dyes into the duplex nucleic acid. As a result, TOTO-1 emitted strong fluorescence and efficient Förster resonance energy transfer (FRET) happened. MicroRNAs miRNA-155, miRNA-182, and miRNA-197, which are significant for the early diagnosis of lung cancer, were simultaneously detected as models. Deviations from spectral crosstalk in the presence of other miRNAs were corrected by mathematical methods. Results demonstrated that, after spectra crosstalk corrections, every miRNA at high or low concentration in plasma was determined accurately in the presence of either high or low concentrations of the other two miRNAs. This new multiplexed assay for miRNAs is promising for clinical diagnosis, prognosis, and therapeutic monitoring of early-stage lung cancer. [ABSTRACT FROM AUTHOR]

A major purpose of exploratory metabolic profiling is for the identification of molecular species that are statistically associated with specific biological or medical outcomes; unfortunately, the structure elucidation process of unknowns is often a major bottleneck in this process. We present here new holistic strategies that combine different statistical spectroscopic and analytical techniques to improve and simplify the process of metabolite identification. We exemplify these strategies using study data collected as part of a dietary intervention to improve health and which elicits a relatively subtle suite of changes from complex molecular profiles. We identify three new dietary biomarkers related to the consumption of peas (N-methyl nicotinic acid), apples (rhamnitol), and onions (N-acetyl-S-(1Z)-propenyl-cysteine- sulfoxide) that can be used to enhance dietary assessment and assess adherence to diet. As part of the strategy, we introduce a new probabilistic statistical spectroscopy tool, RED-STORM (Resolution EnhanceD SubseT Optimization by Reference Matching), that uses 2D J-resolved ¹H NMR spectra for enhanced information recovery using the Bayesian paradigm to extract a subset of spectra with similar spectral signatures to a reference. RED-STORM provided new information for subsequent experiments (e.g., 2D-NMR spectroscopy, solid-phase extraction, liquid chromatography prefaced mass spectrometry) used to ultimately identify an unknown compound. In summary, we illustrate the benefit of acquiring J-resolved experiments alongside conventional 1D ¹H NMR as part of routine metabolic profiling in large data sets and show that application of complementary statistical and analytical techniques for the identification of unknown metabolites can be used to save valuable time and resources. [ABSTRACT FROM AUTHOR]

Effects of medicine on the biomolecular interaction have been given extensive attention in biochemistry and biomedicine because of the complexity of the environment in vivo and the increasing opportunity of exposure to medicine. Herein, the effect of lidocaine on the interactions of C-reactive protein (CRP) with its aptamer and antibody under different temperature was investigated through dynamic force spectroscopy (DFS). The results revealed that lidocaine could reduce the binding probabilities and binding affinities of the CRP-aptamer and the CRP-antibody. An interesting discovery was that lidocaine had differential influences on the dynamic force spectra of the CRP-aptamer and the CRP-antibody. The energy landscape of the CRP-aptamer turned from two activation barriers to one after the treatment of lidocaine, while the one activation barrier in energy landscape of the CRP-antibody almost remained unchanged. In addition, similar results were obtained for 25 and 37 °C. In accordance with the result of molecular docking, the reduction of binding probabilities might be due to the binding of lidocaine on CRP. Additionally, the alteration of the dissociation pathway of the CRP-aptamer and the change of binding affinities might be caused by the conformational change of CRP, which was verified through synchronous fluorescence spectroscopy. Furthermore, differential effects of lidocaine on the interactions of CRP-aptamer and CRP-antibody might be attributed to the different dissociation processes and binding sites of the CRP-aptamer and the CRP-antibody and different structures of the aptamer and the antibody. This work indicated that DFS provided information for further research and comprehensive applications of biomolecular interaction, especially in the design of biosensors in complex systems. [ABSTRACT FROM AUTHOR]

This Article introduces a new computationally efficient noise-tolerant signal processing method, referred to as phased spectrum deconvolution method (ΦSDM), designed for Fourier transform mass spectrometry (FT MS). ΦSDM produces interference-free mass spectra with resolution beyond the Fourier transform (FT) uncertainty limit. With a presumption that the oscillation phases are preserved, the method deconvolves an observed FT spectrum into a distribution of harmonic components bound to a fixed frequency grid, which is several times finer than that of FT. The approach shows stability under noisy conditions, and the noise levels in the resulting spectra are lower than those of the original FT spectra. Although requiring more computational power than standard FT algorithms, ΦSDM runs in a quasilinear time. The method was tested on both synthetic and experimental data, and consistently demonstrated performance superior to the FT-based methodologies, be it across the entire mass range or on a selected mass window of interest. ΦSDM promises substantial improvements in the spectral quality and the speed of FT MS instruments. It might also be beneficial for other spectroscopy approaches which require harmonic analysis for data processing. [ABSTRACT FROM AUTHOR]

Airborne nanoparticles play a key role in climate effects as well as impacting human health. Their small mass and complex chemical composition represent significant challenges for analysis. This work introduces a new ionization method, droplet assisted inlet ionization (DAII), where aqueous droplets are produced from airborne nanoparticles. When these droplets enter the mass spectrometer through a heated inlet, rapid vaporization leads to the formation of molecular ions. The method is demonstrated with test aerosols consisting of polypropylene glycol (PPG), angiotensin II, bovine serum albumin, and the "thermometer" compound p-methoxybenzylpyridinium chloride. High-quality spectra were obtained from PPG particles down to 13 nm in diameter and sampled masses in the low pictogram range. These correspond to aerosol number and mass concentrations smaller than 1000 particles/cm³ and 100 ng/m³, respectively, and a time resolution on the order of seconds. Fragmentation of the thermometer ion using DAII was inlet temperature dependent and similar in magnitude to that observed with a conventional ESI source on the same instrument. DAII should be applicable to other types of aerosols including workplace aerosols and those produced for drug delivery by inhalation. [ABSTRACT FROM AUTHOR]

Interactions between nucleic acids and proteins are critical for many cellular processes, and their study is of utmost importance to many areas of biochemistry, cellular biology, and virology. Here, we introduce a new analytical method based on sedimentation velocity (SV) analytical ultracentrifugation, in combination with a novel multiwavelength detector to characterize such interactions. We identified the stoichiometry and molar mass of a complex formed during the interaction of a West Nile virus RNA stem loop structure with the human T cell-restricted intracellular antigen-1 related protein. SV has long been proven as a powerful technique for studying dynamic assembly processes under physiological conditions in solution. Here, we demonstrate, for the first time, how the new multiwavelength technology can be exploited to study protein-RNA interactions, and show how the spectral information derived from the new detector complements the traditional hydrodynamic information from analytical ultracentrifugation. Our method allows the protein and nucleic acid signals to be separated by spectral decomposition such that sedimentation information from each individual species, including any complexes, can be clearly identified based on their spectral signatures. The method presented here extends to any interacting system where the interaction partners are spectrally separable. [ABSTRACT FROM AUTHOR]

Complex samples benefit from multidimensional measurements where higher resolution enables more complete characterization of biological and environmental systems. To address this challenge, we developed a drift tube-based ion mobility spectrometry-Orbitrap mass spectrometer (IMS-Orbitrap MS) platform. To circumvent the time scale disparity between the fast IMS separation and the much slower Orbitrap MS acquisition, we utilized a dual gate and pseudorandom sequences to multiplex the injection of ions and allow operation in signal averaging (SA), single multiplexing (SM), and double multiplexing (DM) IMS modes to optimize the signal-to-noise ratio of the measurements. For the SM measurements, a previously developed algorithm was used to reconstruct the IMS data. A new algorithm was developed for the DM analyses involving a two-step process that first recovers the SM data and then decodes the SM data. The algorithm also performs multiple refining procedures to minimize demultiplexing artifacts. The new IMS-Orbitrap MS platform was demonstrated by the analysis of proteomic and petroleum samples, where the integration of IMS and high mass resolution proved essential for accurate assignment of molecular formulas. [ABSTRACT FROM AUTHOR]

Centrifugal partition chromatography (CPC) and all countercurrent separation apparatus provide chemists with efficient ways to work with complex matrixes, especially in the domain of natural products. However, despite the great advances provided by these techniques, more efficient ways of analyzing the output flow would bring further enhancement This study describe a hyphenated approach made by coupling NMR with CPC through a hybrid-indirect coupling made possible by using a solid phase extraction (SPE) apparatus intended for high-pressure liquid chromatography (HPLC)- NMR hyphenation. Some hardware changes were needed to adapt the incompatible flow-rates and a reverse-engineering approach that led to the specific software required to control the apparatus. ID ¹HNMR and ¹H--¹H correlation spectroscopy (COSY) spectra were acquired in reasonable time without the need for any solvent-suppression method thanks to the SPE nitrogen drying step. The reduced usage of expensive deuterated solvents from several hundreds of milliliters to the milliliter order is the major improvement of this approach compared to the previously published ones. [ABSTRACT FROM AUTHOR]

Gas chromatography coupled to mass spectrometry (GC/MS) has been a long-standing approach used for identifying small molecules due to the highly reproducible ionization process of electron impact ionization (EI). However, the use of GC-EI MS in untargeted metabolomics produces large and complex data sets characterized by coeluting compounds and extensive fragmentation of molecular ions caused by the hard electron ionization. In order to identify and extract quantitative information on metabolites across multiple biological samples, integrated computational workflows for data processing are needed. Here we introduce eRah, a free computational tool written in the open language R composed of five core functions: (i) noise filtering and baseline removal of GC/MS chromatograms, (ii) an innovative compound deconvolution process using multivariate analysis techniques based on compound match by local covariance (CMLC) and orthogonal signal deconvolution (OSD), (iii) alignment of mass spectra across samples, (iv) missing compound recovery, and (v) identification of metabolites by spectral library matching using publicly available mass spectra. eRah outputs a table with compound names, matching scores and the integrated area of compounds for each sample. The automated capabilities of eRah are demonstrated by the analysis of GC-time-of-flight (TOF) MS data from plasma samples of adolescents with hyperinsulinaemic androgen excess and healthy controls. The quantitative results of eRah are compared to centWave, the peak-picking algorithm implemented in the widely used XCMS package, MetAlign, and ChromaTOF software. Significantly dysregulated metabolites are further validated using pure standards and targeted analysis by GC-triple quadrupole (QqQ) MS, LC-QqQ, and NMR. eRah is freely available at http://CRAN.R-project.org/package=erah. [ABSTRACT FROM AUTHOR]

Atmospheric aerosol nanoparticles play a major role in many atmospheric processes and in particular in the global climate system. Understanding their formation by homogeneous or heterogeneous nucleation as well as their photochemical aging and atmospheric transformation is of utmost importance to evaluate their impact on atmospheric phenomena. Single particle analysis like tip-enhanced Raman spectroscopy (TERS) opens access to a deeper understanding of these nanoparticles. Atmospherically relevant nanoparticles, formed above a simulated salt lake inside an aerosol smog-chamber, were analyzed using TERS. TERS spectra of 11 nanoparticles were studied in detail. First results of TERS on atmospherically relevant aerosol nanoparticles reveal the presence of inorganic seed particles, a chemical diversity of equally sized particles in the nucleation mode, and chemical transformation during photochemical aging. Therefore, single particle analysis by optical near-field spectroscopy such as TERS of atmospheric nanoparticles will significantly contribute to elucidate atmospheric nucleation, photochemical aging, and chemical transformation processes by uncovering single particle based properties. [ABSTRACT FROM AUTHOR]

Gas chromatography using atmospheric pressure chemical ionization coupled to mass spectrometry (GC/APCI-MS) is an emerging metabolomics platform, providing much-enhanced capabilities for structural mass spectrometry as compared to traditional electron ionization (EI)-based techniques. To exploit the potential of GC/APCI-MS for more comprehensive metabolite annotation, a major bottleneck in metabolomics, we here present the novel R-based tool InterpretMSSpectrum assisting in the common task of annotating and evaluating in-source mass spectra as obtained from typical full-scan experiments. After passing a list of mass-intensity pairs, InterpretMSSpectrum locates the molecular ion (M0), fragment, and adduct peaks, calculates their most likely sum formula combination, and graphically summarizes results as an annotated mass spectrum. Using (modifiable) filter rules for the commonly used methoximated-trimethylsilylated (MeOx-TMS) derivatives, covering elemental composition, typical substructures, neutral losses, and adducts, InterpretMSSpectrum significantly reduces the number of sum formula candidates, minimizing manual effort for postprocessing candidate lists. We demonstrate the utility of InterpretMSSpectrum for 86 in-source spectra of derivatized standard compounds, in which rank-1 sum formula assignments were achieved in 84% of the cases, compared to only 63% when using mass and isotope information on the M0 alone. We further use, for the first time, automated annotation to evaluate the purity of pseudospectra generated by different metabolomics preprocessing tools, showing that automated annotation can serve as an integrative quality measure for peak picking/deconvolution methods. As an R package, InterpretMSSpectrum integrates flexibly into existing metabolomics pipelines and is freely available from CRAN (https://cran.r-project.org/). [ABSTRACT FROM AUTHOR]

We describe and illustrate a three-step data-processing approach that enables individual congener groups of chlorinated paraffins (CPs) to be resolved in mass spectra obtained from either of two soft ionization methods: electron capture negative ionization mass spectrometry (ECNI-MS) or atmospheric pressure chemical ionization mass spectrometry (APCI-MS). In the first step, general fragmentation pathways of CPs are deduced from analysis of mass spectra of individual CP congeners. In the second step, all possible fragment ions in the general fragmentation pathways of CPs with 10 to 20 carbon atoms are enumerated and compared to mass spectra of CP mixture standards, and a deconvolution algorithm is applied to identify fragment ions that are actually observed. In the third step, isotope permutations of the observed fragment ions are calculated and used to identify isobaric overlaps, so that mass intensities of individual CP congener groups can be deconvolved from the unresolved isobaric ion signal intensities in mass spectra. For a specific instrument, the three steps only need to be done once to enable deconvolution of CPs in unknown samples. This approach enables congener group-level resolution of CP mixtures in environmental samples, and it opens up the possibility for quantification of congener groups. [ABSTRACT FROM AUTHOR]

We report on the first detailed use of broadband cavity enhanced absorption spectroscopy (BBCEAS) as a detection system for immunoassay. A vertical R ≥ 0.99 optical cavity was integrated with a motorized XY stage, which functioned as a receptacle for 96-well microtiter plates. The custom-built cavity enhanced microplate reader was used to make measurements on a commercially available osteocalcin sandwich ELISA kit. A 30-fold increase in path length was obtained with a minimum detectable change in the absorption coefficient, αmin(t), of 5.3 × 10-5 cm-1 Hz-1/2. This corresponded to a 39-fold increase in the sensitivity of measurement when directly compared to measurements in a conventional microplate reader. Separate measurements of a standard STREP-HRP colorimetric reaction in microtiter plates of differing optical quality produced an increase in sensitivity of up to 115-fold compared to a conventional microplate reader. The sensitivity of the developed setup compared favorably with previous liquid-phase cavity enhanced studies and approaches the sensitivity of typical fluorometric ELISAs. It could benefit any biochemical test which uses single pass absorption as a detection method, through either the label free detection of biologically important molecules at lower concentrations or the reduction in the amount of expensive biochemicals needed for a particular test, leading to cheaper tests. [ABSTRACT FROM AUTHOR]

A real-time method to observe both the structural and the electronic configuration of an organic molecule during deposition is reported for the model system of pentacene on graphite. Structural phase transition of the thin films as a function of coverage is monitored by using in situ angle resolved photoemission spectroscopy (ARPES) results to observe the change of the electronic configuration at the same time. A photoemission theory that uses independent atomic center approximations is introduced to identify the molecular orientation from the ARPES technique. This study provides a practical insight into interpreting ARPES data regarding dynamic changes of molecular orientation during initial growth of molecules on a well-defined surface. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the feasibility of FTIR-ATR spectroscopy coupled with the multivariate numerical methodology for qualitative and quantitative analysis of binary and ternary edible oil mixtures. Four pure oils (extra virgin olive oil, high oleic sunflower oil, rapeseed oil, and sunflower oil), as well as their 54 binary and 108 ternary mixtures, were analyzed using FTIR-ATR spectroscopy in combination with principal component and discriminant analysis, partial least-squares, and principal component regression. It was found that the composition of all 166 samples can be excellently represented using only the first three principal components describing 98.29% of total variance in the selected spectral range (3035-2989, 1170-1140, 1120-1100, 1093-1047, and 930-890 cm-1). Factor scores in 3D space spanned by these three principal components form a tetrahedral-like arrangement: pure oils being at the vertices, binary mixtures at the edges, and ternary mixtures on the faces of a tetrahedron. To confirm the validity of results, we applied several cross-validation methods. Quantitative analysis was performed by minimization of root-mean-square error of cross-validation values regarding the spectral range, derivative order, and choice of method (partial least-squares or principal component regression), which resulted in excellent predictions for test sets (R2 > 0.99 in all cases). Additionally, experimentally more demanding gas chromatography analysis of fatty acid content was carried out for all specimens, confirming the results obtained by FTIR-ATR coupled with principal component analysis. However, FTIR-ATR provided a considerably better model for prediction of mixture composition than gas chromatography, especially for high oleic sunflower oil. [ABSTRACT FROM AUTHOR]

Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) is a powerful tool to study protein folding and to map the interfaces between interacting proteins. The most commonly used cross-linkers in CXMS are BS3 and DSS, which have similar structures and generate the same linkages between pairs of lysine residues in spatial proximity. However, there are cases where no cross-linkable lysine pairs are present at certain regions of a protein or at the interface of two interacting proteins. In order to find the cross-linkers that can best complement the performance of BS3 and DSS, we tested seven additional cross-linkers that either have different spacer arm structures or that target different amino acids (BS2G, EGS, AMAS, GMBS, Sulfo-GMBS, EDC, and TFCS). Using BSA, aldolase, the yeast H/ACA protein complex, and E. coli 70S ribosomes, we showed that, in terms of providing structural information not obtained through the use of BS3 and DSS, EGS and Sulfo-GMBS worked better than the other cross-linkers that we tested. EGS generated a large number of cross-links not seen with the other amine-specific cross-linkers, possibly due to its hydrophilic spacer arm. We demonstrate that incorporating the cross-links contributed by the EGS and amine-sulfhydryl cross-linkers greatly increased the accuracy of Rosetta in docking the structure of the yeast H/ACA protein complex. Given the improved depth of useful information it can provide, we suggest that the multilinker CXMS approach should be used routinely when the amount of a sample permits. [ABSTRACT FROM AUTHOR]

Complementing the demand for effective crime reduction measures are the increasing availability of commercial forensic "taggants", which may be used to physically mark an object in order to make it uniquely identifiable. This study explores the use of a novel "peptide coding" reagents to establish evidence of contact transfer during criminal activity. The reagent, containing a fluorophore dispersed within an oil-based medium, also includes a unique synthetic peptide sequence that acts as a traceable "code" to identify the origin of the taggant. The reagent is detectable through its fluorescent properties, which then allows the peptide to be recovered by swabbing and extracted for electrospray ionization-mass spectrometry (ESI-MS) analysis via a simple liquid-liquid extraction procedure. The performance of the reagent in variable conditions that mimic the limits of a real world use are investigated. [ABSTRACT FROM AUTHOR]

This study describes a novel method for single-cell analysis and lipid profiling by combining drop-on-demand inkjet cell printing and probe electrospray ionization mass spectrometry (PESI-MS). Through inkjet sampling of a cell suspension, droplets with single cells were generated, precisely dripped onto a tungsten-made electrospray ionization needle, and immediately sprayed under a high-voltage electric field. Lipid fingerprints of single cells were obtained by a mass spectrometry (MS) detector. A homemade magnetic stirring device was applied to the cell suspension reservoir, which controlled the homogeneous distribution of cells in liquid and improved the single-cell-droplet percentage by 43.8%. Eight types of single cells were screened in our platform and further differentiated by principal component analysis based on cellular surface phospholipids. Thus, this study successfully provides a facile method for the direct MS profiling of single-cell lipids by PESI-MS. [ABSTRACT FROM AUTHOR]

A new sheathless transient capillary isotachophoresis (CITP)/capillary zone electrophoresis (CZE)-MS interface, based on a commercially available capillary with an integrated metal-coated ESI emitter, was developed in this study aiming at overcoming the reproducibility and ruggedness problems suffered to a certain degree by almost all the available CE-MS interfaces, and pushing the CE-MS technology suitable for routine sample analysis with high sensitivity. The new CITP/CZE-MS interface allows the electric contact between ESI voltage power supply and the CE separation liquid by using a conductive liquid that comes in contact with the metal-coated surface of the ESI emitter, making it a true sheathless CE-MS interface. Stable electrospray was established by avoiding the formation of gas bubbles from electrochemical reaction inside the CE capillary. Crucial operating parameters, such as sample loading volume, flow rate, and separation voltage, were systematically evaluated for their effects on both CITP/CZE separation efficiency and MS detection sensitivity. Around one hundred CITP/CZE-MS analyses can be easily achieved by using the new sheathless CITP/CZE interface without a noticeable loss of metal coating on the ESI emitter surface, or degrading of the ESI emitter performance. The reproducibility in analyte migration time and quantitative performance of the new interface was experimentally evaluated to demonstrate a LOQ below 5 attomole. [ABSTRACT FROM AUTHOR]

Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) allows the correlation between precursor and fragment ions in tandem mass spectrometry without the need to isolate the precursor ion beforehand. 2D FT-ICR MS has been optimized as a data-independent method for the structural analysis of compounds in complex samples. Data processing methods and denoising algorithms have been developed to use it as an analytical tool. In the present study, the capabilities of 2D FT-ICR MS are explored with a tryptic digest of cytochrome c with both ECD and IRMPD as fragmentation modes. The 2D mass spectra showed useful fragmentation patterns of peptides over a dynamic range of almost 400. By using a quadratic calibration, fragment ion peaks could be successfully assigned. The correlation between precursor and fragment ions in the 2D mass spectra was more accurate than in MS/MS spectra after quadrupole isolation, due to the limitations of quadrupole isolation. The use of the second dimension allowed for successful fragment assignment from precursors that were separated by only m/z 0.0156. The resulting cleavage coverage of cytochrome c almost matched data provided by high-resolution FT-ICR MS/MS analysis, but the 2D FT-ICR MS method required only one experimental scan. [ABSTRACT FROM AUTHOR]

Electrochemical or faradaic impedance spectroscopy (EIS) using the ferri/ferrocyanide couple as a redox probe at gold working electrodes was evaluated with respect to its ability to monitor consecutive surface modification steps. As a model reaction, the reversible hybridization and dehybridization of DNA was studied. Thiol-modified single stranded DNA (ssDNA, 20 bases, capture probe) was chemisorbed to a gold electrode and treated with a solution of short thiols to release nonspecifically adsorbed DNA before hybridization with complementary ssDNA (20 bases, target) was carried out. Reversible dehybridization was achieved by intense rinsing with pure water. The experimental procedures were optimized by kinetic surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation (QCM-D) measurements to maximize the increase in reflectivity or decrease in frequency upon hybridization before hybridization/dehybridization was also monitored by EIS. In contrast to SPR and QCM-D, repeatable EIS measurements were not possible at first. Combined SPR/EIS and QCM-D/EIS measurements revealed that during EIS the gold surface is seriously damaged due to the presence of CN- ions, which are released from the ferri/ferrocyanide redox probe. Even at optimized experimental conditions, etching the gold electrodes could not be completely suppressed and the repeatability of the EIS measurements was limited. In three out of four experimental runs, only two hybridization/dehybridization steps could be monitored reversibly by EIS. Thereafter etching the gold electrode significantly contributed to the EIS spectra whereas the QCM-D response was still repeatable. Hence great care has to be taken when this technique is used to monitor surface modification at gold electrodes. [ABSTRACT FROM AUTHOR]

This work demonstrates the ability to detect changes in both quantity and spatial distribution of human stratum corneum (SC) lipids from samples collected in vivo. The SC functions as the predominant barrier to the body, protecting against the penetration of xenobiotic substances. Changes to the SC lipid composition have been associated with barrier impairment and consequent skin disorders, and it is therefore important to monitor and quantify changes to this structure. This work demonstrates the first reported use of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to assess physiological changes to human SC as a function of depth. This technique provides exceptional sensitivity and chemical specificity, allowing analysis of single tape stripped samples taken from volunteers. Using this methodology we were able to successfully identify chemical differences in human SC resulting from both intrinsic and extrinsic (photo) aging. Samples were collected from women of two age groups (under 27 and postmenopausal) and from two body sites with varying UV exposure (inner forearm and dorsal hand), and differences were identified using multivariate data analysis. The key finding was the significant aged-related increase and change in spatial distribution of the sterol cholesterol sulfate, a membrane stabilizing lipid. Significant changes in the prevalence of both lignoceric acid (C24:0) and hexacosanoic acid (C26:0) were also observed. This work describes previously unreported age-related chemical changes to human SC, providing an insight into aging mechanisms which may improve the design of both pharmaceutical and cosmetic topical products. [ABSTRACT FROM AUTHOR]

Product identification is a critical and required analysis for biotheraputics. In addition to regulatory requirements for identity testing on final drug products, in-process identity testing is implemented to reduce business risks associated with fill operations and can also be used as a tool against counterfeiting. Biotherapeutics, in particular monoclonal antibodies, represent a challenging cohort for identity determination because of their similarity in chemical structure. Traditional methods used for product identification can be time and labor intensive, creating a need for quick, inexpensive and reliable methods of drug identification. Here, driven by its molecular-specific and nonperturbative nature, we present Raman spectroscopy as an alternate analytical tool for identity testing. By exploiting subtle differences in vibrational modes of the biologics, we have developed partial least-squares-discriminant analysis derived decision algorithms that offer excellent differentiation capability using spontaneous Raman spectra as well as label-free plasmon-enhanced Raman spectra. Coupled with the robustness to spurious correlations due to its high information content, our results highlight the potential of Raman spectroscopy as a powerful method for rapid, on-site biotherapeutic product identification. [ABSTRACT FROM AUTHOR]