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The first use of statistical correlation spectroscopy to extract chemical information from 2D-HSQC spectra, termed HSQC correlation spectroscopy (HSQCcos), is reported. HSQCcos is illustrated using heparin, a heterogeneous polysaccharide, whose diverse composition causes signals in HSQC spectra to disperse. HSQCcos has been used to probe the chain modifications that cause this effect and reveals hitherto unreported structural details. An interesting finding was that the signal for position 2 of trisulfated glucosamine [N-, 3-O-, and 6-O-sulfated] (A*) is bifurcated, owing to the presence of A* residues in both the "normal" antithrombin binding site and also at the nonreducing end of the molecule, which is reported in intact heparin for the first time. The method was also applied to investigating the environment around other rare sequences/disaccharides, suggesting that the disaccharide; 2-O-sulfated iduronic acid linked to 6-O-sulfated N-glucosamine, which contains a free amine at position 2, is adjacent to the heparin linkage region. HSQCcos can extract chemically related signals from information-rich spectra obtained from complex mixtures such as heparin. [ABSTRACT FROM AUTHOR]

A Fourier transform infrared (FT-IR) spectroscopy study on the entire Italian postage stamps production is presented in this work. Crossing ISO years of issues from the unification of Italy until today, a time line of the major components constituting the stamps has been defined, based on the wide spectral database built on the basis of the numerous analyzed ezemplars. Even though it is easy to find reports about stamps' issues history, information arising from these investigations contributes to throw light upon the substances incorporated in the stamps, which could be described as hybrid or composite materials (a sort of undisclosed or hidden story). As a result of the whole spectra acquired in attenuated total reflectance (ATR) mode, changes in paper composition showed the transition from the protein sizing glue to starch sizing; also the employment of kaolin varied through time. First it was used as the extender in the pigment-medium mixture, and finally it constituted the coating on the stamp surface. Also the chemical composition of the adhesive gum on the rear side of stamps has been subjected to modifications, as well as the front side. The earliest back glue was a protein-based adhesive; then it was replaced by gum arabic first and by poly(vinyl acetate) (PVAC) later. FT-lit spectroscopy, supported by the detailed database developed, has been applied, for the first time, in the very useful detection of two counterfeit samplew a fake of the famous Gronchi Rosa, issued in 1961, and a regunimed 2 cent red stamp, issued in 1865. The information held in the whole spectral data has been selected and employed in the principal component analysis (PCA) statistical analysis. [ABSTRACT FROM AUTHOR]

The discovery of activation state dependent kinase inhibitors, which bind specifically to the inactive conformation of the protein, is considered to be a promising pathway to improved cancer treatments. Identifying such inhibitors is challenging, however, because they can have Kd values similar to molecules known to inhibit kinase function by interacting with the active form. Further, while inhibitor induced changes within the kinase tertiary structure are significant, few technologies are able to correctly assign inhibitor binding modes in a high-throughput fashion based exclusively on protein-inhibitor complex formation and changes in local protein structure. We have developed a new assay, using ion mobility-mass spectrometry, capable of both rapidly detecting inhibitor binding and classifying the resultant kinase binding modes. Here, we demonstrate the ability of our approach to classify a broad set of kinase inhibitors, using micrograms of protein, without the need for protein modification or tagging. [ABSTRACT FROM AUTHOR]

Intermodulation is used for the analysis of the nonlinear behavior of electrochemical and electronic systems. As a matter of fact, different than the passive elements, electrochemical systems have a highly nonlinear character, which can be used to obtain information on the reaction mechanism and structure of the double layer. The setup for measuring and analyzing the intermodulated sidebands is discussed in detail, using a commercial Schottky diode as the ideal system. A general intermodulated differential immitance spectroscopy technique was consequently defined as the analysis of the variation of the immittance elements as a function of the stimulus frequency, and its transfer function was called differential immittance spectrum. Through a simple model, it was possible to precisely calculate the flat band voltage and the doping level of the Schottky diode from a single differential immittance spectrum. The differential immitance spectra of a dummy cell containing passive elements demonstrated the resolution limits of the technique. [ABSTRACT FROM AUTHOR]

Thin-layer polonium (Po) sources for alpha spectrometry counting can be rapidly prepared using copper sulfide (CuS) microprecipitation. Polonium was coprecipitated with CuS, filtered onto Eichrom Resolve filters, and counted. This simple procedure is faster, cheaper, and more convenient than traditional spontaneous plating on metallic discs, and similar yields were obtained (80-90%). The experimental conditions for the microprecipitation technique were optimized (0.05 mg of Cu2+ in 10 mL of 1 M HCl); these compare advantageously with conventional preparation and purification procedures for polonium samples (0.1 to 1 M HCl). The results showed that the most likely potential radionuclide interferences (Ra, Th, U, Np, Pu, and Am) for long-lived polonium isotopes (208Po, 209Po, 210Po) are effectively removed during processing. The effects of several transition metals (Cu2+, Ag+, Fe3+, Fe2+, Pb2+, and Ni2+) on the yield and the resolution of the alpha peaks obtained were also assessed. Little interference was found, demonstrating the versatility of the present microprecipitation technique for environmental and biological matrices. The procedure has been successfully applied to different amounts of 210Po using 209Po as a yield tracer. [ABSTRACT FROM AUTHOR]

We present a capillary photoionization (CPI) method for mass spectrometric (MS) analysis of liquid and gaseous samples. CPI utilizes a heated transfer capillary with a vacuum ultraviolet transparent MgF2 window, through which vacuum UV light (10 eV) from an external source enters the capillary. The liquid or gaseous sample, together with dopant, is introduced directly into the heated transfer capillary between the atmosphere and the vacuum of the MS. Since the sample is vaporized and photoionized inside the capillary, ion transmission is maximized, resulting in good overall sensitivity for nonpolar and polar compounds. As in atmospheric pressure photoionization, ionization in CPI occurs either by proton transfer or by charge exchange reactions. The feasibility of CPI was demonstrated with selected nonpolar and polar compounds. A particular advantage of CPI is that it enables the analysis of nonvolatile and nonpolar compounds in liquid samples with high ionization efficiency. This is not possible with existing capillary ionization methods. The performance of CPI as an interface between GC and MS and its applicability for the analysis of steroids in biological samples are also demonstrated. The GC-CPI-MS method shows good chromatographic resolution, linearity (R² > 0.993), limits of detection (LOD) in the range of 2-6 pg/mL and repeatability of injection with relative standard deviations of 4-15%. [ABSTRACT FROM AUTHOR]

Organic depth profiling using secondary ion mass spectrometry (SIMS) provides valuable information about the three-dimensional distribution of organic molecules. However, for a range of materials, commonly used cluster ion beams such as C60n+ do not yield useful depth profiles. A promising solution to this problem is offered by the use of nitric oxide (NO) gas dosing during sputtering to reduce molecular cross-linking. In this study a C602+ ion beam is used to depth profile a polystyrene film. By systematically varying NO pressure and sample temperature, we evaluate their combined effect on organic depth profiling. Profiles are also acquired from a multilayered polystyrene and polyvinylpyrrolidone film and from a polystyrene/polymethylmethacrylate bilayer, in the former case by using an optimized set of conditions for C602+ and, for comparison, an Ar2000+ ion beam. Our results show a dramatic improvement for depth profiling with C602+ using NO at pressures above 10-6 mbar and sample temperatures below -75 °C. For the multilayered polymer film, the depth profile acquired using C602+ exhibits high signal stability with the exception of an initial signal loss transient and thus allows for successful chemical identification of each of the six layers. The results demonstrate that NO dosing can significantly improve SIMS depth profiling analysis for certain organic materials that are difficult to analyze with C60n+ sputtering using conventional approaches/conditions. While the analytical capability is not as good as large gas cluster ion beams, NO dosing comprises a useful low-cost alternative for instruments equipped with C60n+ sputtering. [ABSTRACT FROM AUTHOR]

Currently, the vast majority of complex carbohydrates are characterized using mass spectrometry (MS)-based techniques. Measuring the molecular mass of a sugar, however, immediately poses a fundamental problem: entire classes of the constituting monosaccharide building blocks exhibit an identical atomic composition and, consequently, also an identical mass. Therefore, carbohydrate MS data can be highly ambiguous and often it is simply not possible to clearly assign a particular molecular structure. A promising approach to overcome the above-mentioned limitation is to implement an additional gas-phase separation dimension using ion mobility spectrometry (IMS), which is a method in which molecules of identical mass and structure but different structure can be separated according to their shape and collision cross section (CCS). With the emergence of commercially available hybrid ion mobility-mass spectrometry (IM-MS) instruments in 2006, IMS technology became readily available. Because of the nonhomogeneous, traveling wave (TW) field utilized in these instruments, however, CCS values currently cannot be determined directly from the drift times measured. Instead, an external calibration using compounds of known CCS and similar molecular identity is required. Here, we report a calibration protocol for TW IMS instruments using a series of sodiated N-glycans that were released from commercially available glycoproteins using an easy-to-follow protocol. The underlying CCS values were determined using a modified Synapt HDMS instrument with a linear drift tube, which was described in detail previously. Our data indicate that, under in-source fragmentation conditions, only a few glycans are required to obtain a TW IMS calibration of sufficient quality. In this context, however, the type of glycan was shown to be of tremendous importance. Furthermore, our data clearly demonstrate that carbohydrate isomers with identical mass but different conformation can be distinguished based on their CCS when all the associated errors are taken into account. [ABSTRACT FROM AUTHOR]

In humans, conjugation with glucuronic acid is the most important phase II metabolic reaction of steroidal compounds. Glucuronoconjugated metabolites have been conventionally studied by using β-glucuronidase enzymes to release the phase I metabolites. It is well-known that hydrolysis with β-glucuronidase presents some limitations that may result in the underestimation of some conjugates. The aim of the present work was to develop and to evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS) scan methods for the open detection of steroid glucuronides in urine samples. The mass spectrometric behavior of thirteen representative steroid glucuronides, used as model compounds, was studied. Characteristic ionization and collision induced dissociation behaviors were observed depending on the steroid glucuronide structure. Neutral loss (NL of 176, 194, 211, and 229 Da) and precursor ion (PI of m/z 141, 159, and 177, in positive mode and m/z 75, 85, and 113, in negative mode) scan methods were evaluated. The NL scan method was chosen for the open detection of glucuronoconjugated steroids due to its sensitivity and the structural information provided by this method. The application of the NL scan method to urine samples collected after testosterone (T) undecanoate administration revealed the presence of two T metabolites which remain conjugated as glucuronides after an enzymatic hydrolysis of the urine. 3α,6β-Dihydroxy-5α-androstan-17-one (6β-hydroxyandrosterone) glucuronide and 3α,6β-dihydroxy-5β-androstan-17-one (6β-hydroxyetiocholanolone) glucuronide were established as the structures for these metabolites, by comparing the structure of the steroids released after chemical hydrolysis with reference materials. An increase of 50-300-fold of these metabolites after oral administration of T undecanoate was observed, proving that their determination can be useful in the doping control field. Moreover, these results exemplify that significant information might be missed, unless direct methods for the determination of steroid glucuronides are employed. [ABSTRACT FROM AUTHOR]

The present work demonstrates, for the first time, the application of the mass spectrometric kinetic method for quantitative chiral purity determination by automatic flow-injection MS/MS. The particular compound analyzed is GSK2251052A, a novel boron-containing systemic antibiotic for the treatment of multidrug-resistant Gram-negative bacterial infections. Chiral recognition and quantitation of GSK2251052A was achieved based on the competitive dissociation kinetics of the CuII-bound trimeric complex [CuII(A)(ref*)2-H]+ (A = GSK2251052A or its R-enantiomer, ref* = l-tryptophan) that gives rise to CuII-bound dimeric complexes. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity, characteristic of the kinetic method, allow chiral purity determination of pharmaceutical compounds during enantioselective synthesis. By using flow-injection MS/MS, enantiomeric quantitation of GSK2251052A by the kinetic method proved to be fast (2 min for analysis of each sample) and to have accuracy comparable to chiral LC-MS/MS and LC-UV methods as well as the method using chiral derivatization followed by LC-MS/MS analysis. This flow-injection MS/MS method represents an alternative approach to commonly used chromatographic techniques as a means of chiral purity determination and is particularly useful for rapid screening of chiral drugs during pharmaceutical development. [ABSTRACT FROM AUTHOR]

One-dimensional 1H NMR spectra are widely used for metabolic profiling. Such data sets often contain hundreds or thousands of spectra, which typically have variation in their sample chemistry, which leads to chemical shift variation "positional noise". This is a severe problem for metabolite quantification and data analysis, as peak integrals do not necessarily correspond across all spectra in a set. Various alignment algorithms have been developed to address this problem, but different studies have taken different approaches to evaluating the performance of NMR alignment routines and can be subjective or rely on an arbitrary cutoff. Furthermore, most alignment approaches completely fail to deal with peaks that overlap. We adopt the simple and robust method of ordering spectra with respect to an internally varying peak and use this to compare different alignment algorithms. Furthermore, we use the information from this procedure to help improve a Bayesian approach to automated peak deconvolution by restricting the prior probability distribution of the peak position in a model-free manner and compare the performance to manual peak deconvolution and to binning. This combination of spectral ordering and compound deconvolution improved the quality of the data for quantitative metabolomics. [ABSTRACT FROM AUTHOR]

We describe a micro-Raman setup allowing for efficient resonance Raman spectroscopy (RRS), i.e., mapping of Raman spectra as a function of tunable laser excitation wavelength. The instrument employs angle-tunable bandpass optical filters which are integrated into software-controlled Raman and laser cleanup filter devices. These automatically follow the excitation laser wavelength and combine tunability with high bandpass transmission as well as high off-band blocking of light. Whereas the spectral intervals which can be simultaneously acquired are bandpass limited to~350 cm-1, they can be tuned across the spectrum of interest to access all characteristic Raman features. As an illustration of performance, we present Raman mapping of single-walled carbon nanotubes (SWNTs): (i) in a small volume of water-surfactant dispersion as well as (ii) after deposition onto a substrate. A significant improvement in the acquisition time (and efficiency) is demonstrated compared to previous RRS implementations. These results may help to establish (micro) Raman spectral mapping as a routine tool for characterization of SWNTs as well as other materials with a pronounced resonance Raman response in the visible-near-infrared spectral region. [ABSTRACT FROM AUTHOR]

The article looks at analytical chemistry methods for the study of abnormal protein and peptide aggregation in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Aspects of protein aggregation in such diseases include amyloid fibril formation, protein folding, protein conformation, and self-assembly. Various analytical methods are explored, such as size exclusion chromatography protein separation, spectroscopy, and immunochemical assays.

Many membrane proteins are proposed to work as oligomers; however, the conclusion is sometimes controversial, as for ß2-adorenergic receptor (ß2AR), which is one of the best-studied family A G-protein-coupled receptors. This is due to the lack of methods for easy and precise detection of the oligomeric state of membrane proteins on living cells. Here, we show that a combination of the coiled-coil tag–probe labeling method and spectral imaging enable a stoichiometric analysis of the oligomeric state of membrane proteins on living cells using monomeric, dimeric, and tetrameric standard membrane proteins. Using this method, we found that ß2ARs do not form constitutive homooligomers, while they exhibit their functions such as the cyclic adenosine 5'-monophosphate (cAMP) signaling and internalization upon agonist stimulation. [ABSTRACT FROM AUTHOR]

The development of spectroscopic and electrochemical devices that can accommodate very small samples is of considerable importance to many areas of science and technology. We report here on the design and characteristics of a simple apparatus for the electrochemical and spectroelectrochemical analysis of submicroliter aqueous samples. The device is easily assembled from common laboratory materials and equipment, utilizing a bifurcated fiber-optic probe, standard disk electrodes of millimeter dimensions, and a polymer electrolyte film salt bridge to enable the analysis of nanoliter-scale sample volumes in a thin-layer configuration. Excellent performance has been demonstrated via measurements on aqueous ferricyanide solutions using sample volumes as low as 20 nL. [ABSTRACT FROM AUTHOR]

The mass spectrometric (MS) complexity associated with the quasi-continuous distribution of mass and charge (m, z) of electrosprayed industrial polymers may be moderated by use of ion mobility spectrometry (IMS) and MS in series. However, when the high charge levels typical of polar polymers stretch the gas phase ions into linear configurations, the mobility Z tends to be closely correlated with m/z, and IMS-MS does not yield spectra more readily interpretable than pure MS spectra. Here we note that the usual high charge states observed in the ESI of polyethylene glycol (PEG) arise because the stretched gas phase chain is able to strongly bind solution cations. We weaken this binding and therefore moderate the charge level by electrospraying in negative mode (NESI). This produces exclusively globular gas phase ions. IMS-MS then readily separates into distinct bands the different z-states, enabling an unambiguous assignment of all ions and simplifying the determination of mass distributions ƒz(m) for each charge state. The measured probability pz(m) that a polymer ion of given mass m will carry z charges spans a surprisingly narrow z range, each mass being present at most in two charge states. PEG ions of a given charge state z become unstable at a critical mass, below which they shed just one elementary charge, evidently by ion evaporation. We argue that NESI-IMS-MS offers significant analytical advantages over alternative methods previously demonstrated, particularly at increasing masses, when individual ion peaks can no longer be discerned. [ABSTRACT FROM AUTHOR]

Using a two-dimensional (2D) correlation analysis technique to determine the sequential order of physical or chemical events has received keen interests in the past ten years. However, our continuous work demonstrates that the sequential order of events determined by the "sequential order" rules of this technique may lead to ambiguous or even wrong conclusions, because the physical significance of the sequential order in generalized 21) correlation analysis is neither well-defined nor meaningful in general situations, and the word "occur" used in the "sequential order" rules may easily give rise to ambiguity. In contrast to the integrated sequential order derived from periodic changes as in mechanical perturbation based 21) correlation infrared spectroscopy, there is a local/chronological sequential order for nonperiodic changes in general situations. The current work shows that the integrated sequential order in 2D correlation analysis is a reflection of the sequential order of the phases, i.e., phase sequence/difference. The integrated sequential order may indicate the relative state of two events (one event occurs/exists before or after the other one) according to a specific reference, only if both are obtained under the same frequency for periodic changes or even speeds for nonperiodic changes in general situations. The integrated sequential order may not always be able to reveal whether one event occurs/happens before or after another one for nonperiodic changes in terms of timings of happenings. For nonperiodic changes, the integrated sequential order is not so meaningful and must be replaced by the local/chronological sequential order. To judge whether one event occurs/happens before or after another one for two nonperiodic changes in general situations, the original spectral intensity changes must be verified to determine if a chronological/local sequential order exists between two events. [ABSTRACT FROM AUTHOR]

An essential part to calibration is establishing the analyte calibration reference samples. These samples must characterize the sample matrix and measurement conditions (chemical, physical, instrumental, and environmental) of any sample to be predicted. Calibration usually requires measuring spectra for numerous reference samples in addition to determining the corresponding analyte reference values. Both tasks are typically time-consuming and costly. This paper reports on a method named pure component Tikhonov regularization (PCTR) that does not require laboratory prepared or determined reference values. Instead, an analyte pure component spectrum is used in conjunction with nonanalyte spectra for calibration. Nonanalyte spectra can be from different sources including pure component interference samples, blanks, and constant analyte samples. The approach is also applicable to calibration maintenance when the analyte pure component spectrum is measured in one set of conditions and nonanalyte spectra are measured in new conditions. The PCTR method balances the trade-offs between calibration model shrinkage and the degree of orthogonality to the nonanalyte content (model direction) in order to obtain accurate predictions. Using visible and near-infrared (NIR) spectral data sets, the PCTR results are comparable to those obtained using ridge regression (RR) with reference calibration sets. The flexibility of PCTR also allows including reference samples if such samples are available. [ABSTRACT FROM AUTHOR]

A new method is described for measuring the short-wave infrared (SWIR) emission wavelengths of numerous individual nanoparticles without using a dedicated spectrometer. Microscope objectives designed for use at visible wavelengths often show severe axial chromatic aberration in the SWIR. This makes coplanar objects emitting at different SWIR wavelengths appear to focus at different depths. After this aberration has been calibrated for a particular objective lens, the depth at which an emissive nanoparticle appears brightest and best focused can be used to deduce its peak emission wavelength. The method is demonstrated using a dilute, structurally polydisperse sample of single-walled carbon nanotubes deposited onto a microscope slide. Discrete emission centers in this sample have different peak wavelengths corresponding to specific nanotube structural species. A set of images was recorded at stepped focus settings and analyzed to find the sharpest focus depth of each nanotube. The chromatic aberration calibration curve converted these depths into peak emission wavelengths with a spectral resolution better than 3 nm, allowing identification of each nanotube's structure. Chromatic aberration spectroscopy is a practical tool for using existing microscopic equipment to extract significant spectral information on coplanar nanoparticle samples that emit or scatter light. [ABSTRACT FROM AUTHOR]

Droplet-based microfluidic systems have become a very powerful tool to miniaturize chemical and biological reactions. However, droplet content analysis remains challenging and relies almost exclusively on optical methods such as fluorescence spectroscopy. Hence, labeling of the analyte is typically required which impedes a more universal applicability of microdroplets. Here we present a novel interface coupling droplet microfluidics and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for label-free content analysis of single droplets. Nanoliter aqueous droplets immersed in perfluorinated oil are created in a microfluidic T-junction, transferred into a capillary, and deposited on a high-density microarray MALDI plate mounted on a motorized xy-stage. The fully automated system is robust and reliable due to two unique features. First, a simple optical droplet detection system is used to synchronize stage movement and exit of droplets from the capillary. Second, the microarray plate contains an array of over 26?000 hydrophilic spots within a hydrophobic coating, each spot acting as a recipient to confine the droplets and to prevent cross-contamination. The MALDI matrix can also be applied using our system by spotting matrix droplets on the microarray in a separate run. To demonstrate the potential of our system, we studied the enzymatic cleavage of angiotensin I by angiotensin converting enzyme and monitored the increasing concentration of the product angiotensin II over time. The interface provides a robust and fully automated method for rapid label-free and information-rich content analysis of single droplets. With the high number of droplets per plate, this method is particularly suitable for high-throughput screening applications. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) are the most widely investigated delivery systems for nucleic acid-based therapeutics and vaccines. Loading efficiency of nucleic acids may vary with formulation conditions, and it is considered one of the critical quality attributes of LNP products. Current analytical methods for quantification of cargo loading in LNPs often require external standard preparations and preseparation of unloaded nucleic acids from LNPs; therefore, they are subject to tedious and lengthy procedures, LNP stability, and unpredictable recovery rates of the separated analytes. Here, we developed a modeling approach, which was based on locally weighted regression (LWR) of ultraviolet (UV) spectra of unpurified samples, to quantify the loading of nucleic acid cargos in LNPs in-situ . We trained the model to automatically tune the training library space according to the spectral features of a query sample so as to robustly predict the nucleic acid cargo concentration and rank loading capacity with similar performance as the more complicated experimental approaches. Furthermore, we successfully applied the model to a wide range of nucleic acid cargo species, including antisense oligonucleotides, single-guided RNA, and messenger RNA, in varied lipid matrices. The LWR modeling approach significantly saved analytical time and efforts by facile UV scans of 96-well sample plates within a few minutes and with minimal sample preprocessing. Our proof-of-concept study presented the very first data mining and modeling strategy to quantify nucleic acid loading in LNPs and is expected to better serve high-throughput screening workflows, thereby facilitates early-stage optimization and development of LNP formulations.

Axial view liquid electrode plasma-atomic emission spectrometry (axial view LEP-AES) was proposed and fabricated successfully in this work. The emission spectra from Zn, Cd, Pb, Ca, and K were applied for characterization and optimization. Comparing with conventional radial view LEP-AES devices, the newly designed axial view-LEP provided better sensing ability toward trace heavy metals. Moreover, pulsed voltage discharge was found to be advantageous over continuous discharge under the same discharge time for detection. The optimized parameters facilitate the limit of detection to achieve 0.24, 0.051, and 0.85 μg L -1 for Zn, Cd, and Pb, respectively. Furthermore, the axial view LEP-AES possessed excellent reproducibility and good durability. The real sample tests using two different certified reference water samples revealed the great potential of the axial view LEP-AES as a novel practical elemental analysis tool.

Polymeric nanocarriers (PNCs) are versatile drug delivery vehicles capable of delivering a variety of therapeutics. Quantitatively monitoring their uptake in biological systems is essential for realizing their potential as next-generation delivery systems; however, existing quantification strategies are limited due to the challenges of detecting polymeric materials in complex biological samples. Here, we describe a metal-coded mass tagging approach that enables the multiplexed quantification of the PNC uptake in cells using mass spectrometry (MS). In this approach, PNCs are conjugated with ligands that bind strongly to lanthanide ions, allowing the PNCs to be sensitively quantitated by inductively coupled plasma-MS. The metal-coded tags have little effect on the properties or toxicity of the PNCs, making them biocompatible. We demonstrate that the conjugation of different metals to the PNCs enables the multiplexed analysis of cellular uptake of multiple distinct PNCs at the same time. This multiplexing capability should improve the design and optimization of PNCs by minimizing biological variability and reducing analysis time, effort, and cost.

Inflammation is associated with many diseases, so the development of an excellent near infrared fluorescent (NIRF) and photoacoustic (PA) dual-modality probe is crucial for the accurate diagnosis and efficacy evaluation of inflammation. However, most of the current NIRF/PA scaffolds are based on repurposing existing fluorescent dye platforms that exhibit non-optimal properties for both NIRF and PA signal outputs. Herein, we developed a novel dye scaffold QL-OH by optimizing the NIRF and PA signal of classical hemicyanine dyes. Based on this optimized dye, we developed the first NIRF/PA dual-mode carbon monoxide (CO) probe QL-CO for noninvasive and sensitive visualization of CO levels in deep inflammatory lesions in vivo. The novel probe QL-CO exhibited rapid and sensitive NIRF 775 /PA 730 dual activation responses toward CO. In addition, the CO-activated probe QL-CO was successfully used for the diagnosis of inflammation and evaluation of anti-inflammation drug efficacy in living mice though the NIRF/PA dual-mode imaging technology for the first time. More importantly, the probe QL-CO could accurately locate the deep inflammatory lesion tissues (≈1 cm) in mice and obtain 3D PA diagnostic images with deep penetration depth and spatial resolution. Therefore, the new NIRF/PA dual-mode probe QL-CO has high potential for deep-tissue diagnosis imaging of CO in vivo. These findings may provide a new tool and approach for future research and diagnosis of CO-associated diseases.

In many areas, studying photostability or the mechanism of photodegradation is of high importance. Conventional methods to do so can be rather time-consuming, laborious, and prone to experimental errors. In this paper we evaluate an integrated and fully automated system for the study of light-induced degradation, comprising a liquid handler, an irradiation source and exposure cell with dedicated optics and spectrograph, and a liquid chromatography (LC) system. A liquid core waveguide (LCW) was used as an exposure cell, allowing efficient illumination of the sample over a 12 cm path length. This cell was coupled to a spectrograph, allowing in situ absorbance monitoring of the exposed sample during irradiation. The LCW is gas-permeable, permitting diffusion of air into the cell during light exposure. This unit was coupled online to LC with diode array detection for immediate and automated analysis of the composition of the light-exposed samples. The analytical performance of the new system was established by assessing linearity, limit of detection, and repeatability of the in-cell detection, sample recovery and carryover, and overall repeatability of light-induced degradation monitoring, using riboflavin as the test compound. The applicability of the system was demonstrated by recording a photodegradation time profile of riboflavin.

The anthraquinone components of the roots of various species of madder (like Rubia tinctorum L. and Rubia peregrina L.) have been used for millennia as red colorants in textiles, carpets, tapestries, and other objects. To understand the selection and preparation of dyestuffs in various cultures and historical periods, these dyes (mainly alizarin and purpurin) are traditionally analyzed by means of separation methods that require sampling. This contribution focuses on establishing a fast, noninvasive, and in situ analytical procedure based on visible reflectance spectroscopy for the characterization and quantification of anthraquinones in ancient wool yarns. The method was successfully applied to Coptic textiles, and the analytical results are in agreement with prior observations obtained on samples by separation techniques.

In this study, we measure the absolute isotope ratios of ytterbium (Yb) by multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) using an optimized regression model for mass bias correction. A rhenium (Re) reference material (NIST SRM 3143), which has been characterized previously, is selected as a primary calibrator to calibrate the absolute Yb isotope ratios for three Yb materials (GSB, Alfa Yb, and GBW). The three-isotope plot for all collected data indicates that the results of Yb isotope ratios obtained are not affected by any polyatomic interferences and the mass-independent isotopic fractionation. Furthermore, the recalibrated Hf historical isotope ratios by using the absolute Yb isotopic composition obtained in this study for the isobaric interference correction on Hf isotopes are in agreement with the original historical values. This work has further demonstrated the applicability of the regression model for the calibrated measurements of absolute isotope ratios using MC-ICP-MS. The three mono-elemental Yb standard solutions are thus proposed as the reference materials for Yb isotope ratio measurements in environmental and geoscience applications.

Silicon (Si) is promising as a field enhancement material because of its high abundance, low toxicity, and high refractive index. The field enhancement effect intensifies light-matter interactions, which improves photocatalysis, solar cell performance, and sensor sensitivity. To manufacture field enhancement materials on a production scale, the fabrication technique must be simple, cost-effective, fast, and highly reproducible and must produce a high enhancement factor (EF). Herein, we report on an economical and efficient fabrication method for a field enhancement substrate consisting of a two-dimensional Si wire array (2D-SiWA). This substrate was demonstrated as a fluorescence sensor with high sensitivity (EF > 200) and composed of a large area (6.0 mm 2 ). In addition, single wire spectroscopy was used to identify very high reproducibility of the sensor sensitivity in regular regions (97%) and a mixture of regular and irregular regions (87%) of the 2D-SiWA. The large-area Si fluorescence sensor fabrication was cost-effective and rapid and was 50× less expensive, 20×faster, and 60,000×larger than the typical electron beam lithography method.

High-throughput single-cell analysis is critical to elucidate the cell heterogeneity. Recently, droplet microchips using oil/gas phases to generate single-cell encapsulated droplets have been combined with inductively coupled plasma-mass spectrometry (ICP-MS) for determination of trace elements in single cells with a throughput of dozens of cells per min. To improve the sample throughput and avoid the oil phase introduced into ICP-MS, herein, a negative magnetophoresis focusing microchip was established and online-coupled to ICP-MS for single-cell analysis. MCF-7 cells in the paramagnetic salt solution were introduced into the designed focusing microchannel, in which they were focused into a single stream under both the magnetic repulsion force and inertial lift force, and then were introduced into ICP-MS for online single-cell analysis. The important parameters including the chip design, the concentration of the paramagnetic salt solution, flow rate, cell density, and dwell time were optimized. Under the optimal conditions, a high sample throughput of 1390 cells min -1 was obtained. The established online analytical system was applied to study the uptake behaviors of MCF-7 cells for Zn 2+ and ZnO nanoparticles (NPs) at a single-cell level. The single-cell analysis results indicate that MCF-7 cells displayed more remarkable heterogeneity when they were treated with ZnO NPs, and the uptake content of ZnO NPs by MCF-7 cells was less than that of Zn 2+ . Compared with other droplet microdevice-ICP-MS analysis systems, the developed system has the advantages of simple design and fabrication, no organic phase, a high throughput, and a low sample consumption (only 5 μL).

The molecular pathology of breast cancer is challenging due to the complex heterogeneity of cellular subtypes. The ability to directly identify and visualize cell subtype distribution at the single-cell level within a tissue section enables precise and rapid diagnosis and prognosis. Here, we applied mass spectrometry imaging (MSI) to acquire and visualize the molecular profiles at the single-cell and subcellular levels of 14 different breast cancer cell lines. We built a molecular library of genetically well-characterized cell lines. Multistep processing, including deep learning, resulted in a breast cancer subtype, the cancer's hormone status, and a genotypic recognition model based on metabolic phenotypes with cross-validation rates of up to 97%. Moreover, we applied our single-cell-based recognition models to complex tissue samples, identifying cell subtypes in tissue context within seconds during measurement. These data demonstrate "on the spot" digital pathology at the single-cell level using MSI, and they provide a framework for fast and accurate high spatial resolution diagnostics and prognostics.

Criegee intermediates, derived from ozonolysis of alkenes and recognized as key species in the production of nonphotolytic free radicals, play a crucial role in atmospheric chemistry. Here, we present a spectrometer based on synchronized two-color time-resolved dual-comb spectroscopy, enabling simultaneous spectral acquisitions in two molecular fingerprint regions near 2.9 and 7.8 μm. Upon flash photolysis of CH 2 I 2 /O 2 /N 2 gas mixtures, multiple reaction species, involving the simplest Criegee intermediates (CH 2 OO), formaldehyde (CH 2 O), hydroxyl (OH) and hydroperoxy (HO 2 ) radicals are simultaneously detected with microsecond time resolution. The concentration of each molecule can be determined based on high-resolution rovibrational absorption spectroscopy. With quantitative detection and simulation of temporal concentration profiles of the targeted molecules at various conditions, the underlying reaction mechanisms and pathways related to the formation of the HO x radicals, which can be generated from decomposition of initially energized and vibrationally excited Criegee intermediates, are explored. This approach capable of achieving multispectral measurements with simultaneously high spectral and temporal resolutions opens up the opportunities for quantification of transient intermediates and products, thus, enabling elucidation of complex reaction mechanisms.

Plasmon rulers relate the shift of resonance wavelength, λ l , of gold agglomerates to the average distance, s , between their constituent nanoparticles. These rulers are essential for monitoring the dynamics of biomolecules ( e.g., proteins and DNA) by determining their small (<10 nm) coating thickness. However, existing rulers for dimers and chains estimate coating thicknesses smaller than 10 nm with rather large errors (more than 200%). Here, the light extinction of dimers, 7- and 15-mers of gold nanoparticles with diameter d p = 20-80 nm and s = 1-50 nm is simulated. Such agglomerates shift λ l up to 680 nm due to plasmonic coupling, in excellent agreement with experimental data by microscopy, dynamic light scattering, analytical centrifugation, and UV-visible spectroscopy. Subsequently, a new plasmon ruler is derived for gold nanoagglomerates that enables the accurate determination of sub-10 nm coating thicknesses, in excellent agreement also with tedious microscopy measurements.

Cryogenic superconducting tunnel junction (STJ) detectors have the advantage of single-particle sensitivity, high quantum efficiency, low noise, and the ability to detect the time and relative impact energy of deposited ions. This makes them attractive for use in mass spectrometry (MS) and as a form of energy spectrometry. STJ cryodetectors have been coupled to time-of-flight (TOF) mass spectrometers equipped with a matrix-assisted laser desorption ionization (MALDI) source and to an electrospray ionization (ESI) TOF mass spectrometer. Here, a lab-made linear quadrupole ion trap (LIT) mass spectrometer system was coupled to an ESI source and a 16-channel Nb-STJ array with improved readout electronics. The goal was to investigate fundamentals of ESI-generated protein ions by further exploiting the advantage of resolving these ions in a third dimension of the relative energy deposited into the STJs. The proteins equine cytochrome c, bovine carbonic anhydrase, bovine serum albumin, and murine immunoglobulin G were studied using this ESI-LIT-STJ-MS instrument. Multiply charged monomers, multimers, and fragments from metastable ions were resolved from monomer peaks by differences in ion deposition energy even when these ions have the same mass-to-charge ratio as the corresponding monomer. The determination of a fragment mass from metastable decomposition is accomplished without knowing the charge state of the fragment. The average charge state of the multimers is reduced with each addition of a protein which is presumed to be a direct reflection of the surface area available for charging. Multiply charged in-source fragments have also been observed and distinguished in the mass spectrum of carbonic anhydrase by using the differences in the energy deposited in the STJs.

The stable isotopes of hydrogen (δ2H) and oxygen (δ18O) in human urine are measured during studies of total energy expenditure by the doubly labeled water method, measurement of total body water, and measurement of insulin resistance by glucose disposal among other applications. An ultrasensitive laser absorption spectrometer based on off-axis integrated cavity output spectroscopy was demonstrated for simple and inexpensive measurement of stable isotopes in natural isotopic abundance and isotopically enriched human urine. Preparation of urine for analysis was simple and rapid (approximately 25 samples per hour), requiring no decolorizing or distillation steps. Analysis schemes were demonstrated to address sample-to-sample memory while still allowing analysis of 45 natural or 30 enriched urine samples per day. The instrument was linear over a wide range of water isotopes (δ2H = −454 to +1702 ‰ and δ18O = −58.3 to +265 ‰). Measurements of human urine were precise to better than 0.65 ‰ 1σ for δ2H and 0.09 ‰ 1σ for δ18O for natural urines, 1.1 ‰ 1σ for δ2H and 0.13 ‰ 1σ for δ18O for low enriched urines, and 1.0 ‰ 1σ for δ2H and 0.08 ‰ 1σ for δ18O for high enriched urines. Furthermore, the accuracy of the isotope measurements of human urines was verified to better than ±0.81 ‰ in δ2H and ±0.13 ‰ in δ18O (average deviation) against three independent isotope-ratio mass spectrometry laboratories. The ability to immediately and inexpensively measure the stable isotopes of water in human urine is expected to increase the number and variety of experiments which can be undertaken. [ABSTRACT FROM AUTHOR]

One of the fastest developing fields in analytical spectrochemistry in recent years is ambient desorption/ionization mass spectrometry (ADI-MS). This burgeoning interest has been due to the demonstrated advantages of the method: simple mass spectra, little or no sample preparation, and applicability to samples in the solid, liquid, or gaseous state. One such ADI-MS source, the flowing atmospheric-pressure afterglow (FAPA), is capable of direct analysis of solids just by aiming the source at the solid surface and sampling the produced ions into a mass spectrometer. However, direct introduction of significant volumes of liquid samples into this source has not been possible, as solvent loads can quench the afterglow and, thus, the formation of reagent ions. As a result, the analysis of liquid samples is preferably carried out by analyzing dried residues or by desorbing small amounts of liquid samples directly from the liquid surface. In the former case, reproducibility of sample introduction is crucial if quantitative results are desired. In the present study, introduction of liquid samples as very small droplets helps overcome the issues of sample positioning and reduced levels of solvent intake. A recently developed "drop-on-demand" (DOD) aerosol generator is capable of reproducibly producing very small volumes of liquid (∼17 pL). In this paper, the coupling of FAPA-MS and DOD is reported and applications are suggested. Analytes representing different classes of substances were tested and limits of detections were determined. Matrix tolerance was investigated for drugs of abuse and their metabolites by analyzing raw urine samples and quantification without the use of internal standards. Limits of detection below 2 μg/mL, without sample pretreatment, were obtained. [ABSTRACT FROM AUTHOR]

This study describes the relationship between the output signal of the ion mobility spectrometry (IMS) detector and the concentrations of two compounds being simultaneously introduced into the reaction section. Investigations were performed for three pairs of compounds, that is, dimethyl methylphosphonate (DMMP) and acetone, methyl tert-butyl ether (MTBE), and acetone, as well as trimethylamine (TMA) and n-nonylamine (NA). Vapors of the investigated compounds were produced in a two- channel generator with permeation sources and a dilution system based on mass-flow controllers. The generator design and the method of concentration determination are discussed in this paper. It was found that admixture can differently influence detection of an analyte. The presence of acetone does not effect the signal corresponding to dimer ions of DMMP. For pairs MTBE + acetone and TMA + NA characteristic peaks of analyte ions diminish with growing concentration of admixture, however, the detection based on the peak of the asymmetric dimer containing proton-bound molecules of both compounds is effective. For the detection of TMA in the presence of NA, the signal generated by the asymmetric dimer ions is meaningfully higher than the signals of monomer or dimer TMA ions measured without the NA admixture. The course of calibration dependencies was analyzed on the basis of a simple mathematical model of the reaction region. This model provided an estimation of the intensity of the signal for a given ionic species for definite concentration of analyte. [ABSTRACT FROM AUTHOR]

Raman spectra were collected from a 1.25 M aqueous pyridine solution, 100-nm polystyrene film or a trimethyl(phenyl)silane monolayer at a plasmon waveguide interface under total internal reflection (TIR). The plasmon waveguide resonance (PWR) interface consisted of a sapphire prism/49 to 50 nm Au/548 to 630 nm SiO2 and a monolayer, thin film or aqueous analyte. The Raman peak area as a function of incident angle was measured using a 785-nm excitation wavelength, and was compared to the Raman peak area obtained at a sapphire or sapphire/50 nm Au interface. In contrast to measurements at a bare sapphire prism, increased surface sensitivity and signal were obtained from the PWR interface. In contrast to measurements at a bare Au film where only p-polarized incident light generates an enhanced interfacial electric field, plasmon waveguide interfaces enable excitation with orthogonal polarizations using s- or p-polarized incident light. The Raman scatter from a monolayer was recorded at the PWR interface with a signal-to-noise ratio of 5.6 when averaging 3 accumulations with 3 min acquisition times using nonresonant excitation, whereas no signal was recorded from a monolayer at the sapphire interface. The reflected light from the interface enabled the identification of the incident angle where the maximum Raman scatter was produced, and the Raman signal generated at the plasmon waveguide interface was modeled by the enhanced interfacial mean square electric field relative to the incident field. In comparison to the techniques on which this work was based (i.e., PWR spectroscopy, TIR Raman spectroscopy at the prism interface, and surface plasmon resonance (SPR) Raman spectroscopy at the prism/Au interface), chemical specificity was added to PWR spectroscopy, a signal enhancement mechanism was introduced for TIR Raman spectroscopy, and polarization control of the interfacial electric field was added to SPR Raman spectroscopy. [ABSTRACT FROM AUTHOR]

Chemical sensors based on a polymer coated quartz crystal microbalance (QCM) generally present poor molecular selectivity for compounds that contain similar functional groups and possess the same chemical properties. This paper shows for the first time that the selectivity and sensitivity of a poly(methyl methacrylate) (PMMA) based QCM sensor can be significantly enhanced for aromatic hydrocarbons by incorporating a plasticizer into the polymer film. The sensor was fabricated by spin coating PMMA onto a quartz crystal, and the influence of plasticizer type and amount on the response was evaluated. It was shown that the hydrocarbon sensitivity of plasticizer-free PMMA is negligible, while the sensitivity of plasticized PMMA was similar to or in some cases greater relative to highly responsive rubbery polymers such as polyisobutylene (PIB). Detection limits of 4.0, 1.5, 0.4, 0.6, and 0.1 ppm were obtained on a PMMA film containing 25% w/w di(2-ethylhexyl) phthalate for benzene, toluene, ethylbenzene, p-xylene, and naphthalene, respectively. We found that at low plasticizer levels (10% w/w) the PMMA film was more sensitive toward ethylbenzene and p-xylene over naphthalene when compared to a PIB film under similar measurement conditions. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) measurements were performed to understand the sensing mechanism, and these studies confirmed a higher hydrocarbon uptake by PMMA in the presence of plasticizer. Positron annihilation lifetime spectroscopy (PALS) studies detected variations in the free volume properties of the polymer films as a function of plasticizer content. The accessible free volume as measured by PALS was significantly less in the PMMA films compared to the PIB, and this result correlates favorably with differences in the QCM response pattern. The QCM results have been rationalized in terms of free volume theory which is responsible for the higher hydrocarbon diffusion/sorption with increased plasticizer content. [ABSTRACT FROM AUTHOR]

In this study, laser desorption ionization (LDI) coupled to Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS) was applied to study crude oils at the molecular level. Molecular ions were the major type of ion detected by (+) mode, and deprotonated and radical anions were the major ions observed by (-) mode LDI FTICR MS. N1 and hydrocarbon classes were dominant in the class distribution plots obtained by (+) LDI FTICR MS, but other heteroatom classes, including Ox and S1, were abundant in plots obtained by (-) LDI FTICR MS. Detailed analysis of double-bond equivalence (DBE) vs carbon number plots revealed that LDI FTICR MS is more sensitive toward polyaromatic compounds than mono- or dicyclic-aromatic compounds. However, nonaromatic and aromatic O2 compounds could be detected simultaneously. An abundance of nonaromatic O2 compounds (presumably naphthenic acids) are correlated with total acid numbers, but O2 compounds with condensed structures are not. Overall, this study shows that LDI FTICR MS can be successfully used to study crude oils at the molecular level. [ABSTRACT FROM AUTHOR]

Surface patterns were observed on spin-coated poly(bisphenol A decane ether) (BA-C10) films prepared with chloroform and tetrahydrofuran as the solvents. The interior structure of these surface patterns were analyzed using a time-of-flight secondary ion mass spectrometry (ToF-SIMS) equipped with a bismuth cluster source for ion imaging and a C60+ cluster source for depth profiling. For the first time, the surface patterns have been shown to be hollow rather than solid using ToF-SIMS three-dimensional (3D) analysis and optical techniques. Moreover, the microarea depth profiling analysis indicated that the hollow structure was sandwiched between two polymer layers rather than sitting on the substrate. The height of the hollow structure and the thicknesses of the polymer layers above and below the hollow structure were also estimated from the depth profiling results. [ABSTRACT FROM AUTHOR]

We have demonstrated two significant benefits of dynamic surface enhanced raman spectroscopy (DSERS) measurements: removal of instrumental and normal Raman interferences in surface enhanced raman spectroscopy (SERS) spectroscopy and site-selective spectroscopy of adsorbate populations on SERS-active particles. Our first example of shelled nanoparticles at very low concentrations confirmed the benefit of DSERS for removal of an overwhelmingly strong solvent spectral interference. The second benefit, site selection, was demonstrated with 4-mercaptopyridine on bare Au nanoparticles to observe a small population of molecules that were spectroscopically unique from the large population of molecules on the particles. The DSERS spectrum originated from excess variance between a small population of adsorbates on the ensemble of nanoparticles. [ABSTRACT FROM AUTHOR]

A sequential identification approach by two-dimensional (2D) correlation analysis for the identification of a chemical reaction model, activation, and thermodynamic parameters is presented in this paper. The identification task is decomposed into a sequence of subproblems. The first step is the construction of a reaction model with the suggested information by model-free 2D correlation analysis using a novel technique called derivative double 2D correlation spectroscopy (DD2DCOS), which enables one to analyze intensities with nonlinear behavior and overlapped bands. The second step is a model-based 2D correlation analysis where the activation and thermodynamic parameters are estimated by an indirect implicit calibration or a calibration-free approach. In this way, a minimization process for the spectral information by sample–sample 2D correlation spectroscopy and kinetic hard modeling (using ordinary differential equations) of the chemical reaction model is carried out. The sequential identification by 2D correlation analysis is illustrated with reference to the isomeric structure of diphenylurethane synthesized from phenylisocyanate and phenol. The reaction was investigated by FT-IR spectroscopy. The activation and thermodynamic parameters of the isomeric structures of diphenylurethane linked through a hydrogen bonding equilibrium were studied by means of an integration of model-free and model-based 2D correlation analysis called a sequential identification approach. The study determined the enthalpy (ΔH = 15.25 kJ/mol) and entropy (TΔS = 13.20 kJ/mol) of C═O···H hydrogen bonding of diphenylurethane through direct calculation from the differences in the kinetic parameters (δΔH, −TδΔS) at equilibrium in the chemical reaction system. [ABSTRACT FROM AUTHOR]

Broad-band operation is investigated as an alternative means of refractive index detection in an optical ring resonator. In this work, a liquid-core (LCORR, capillary-based) design is used. Optical ring resonators have been recently demonstrated for the detection of a wide variety of analytes, including DNA, viruses, proteins, chemical vapors, and pesticides. In the field of analytical chemistry, much of their value is in the ability to provide enhanced detection of surface analytes in small volumes. Conventional analysis methods that employ a single resonant wavelength and propagation mode are highly dependent on resonator quality. By utilizing the complex and variable response of a multiple-mode resonator and the simultaneous data of more than 40 resonance peaks, impressive results exceeding the single-mode prediction are produced from a very modest quality device. The presented methods also become attractive through the use of inexpensive LED light sources and common UV-vis spectrometers, as well as the ability to also take absorbance measurements without any physical configuration changes. Complex interference spectra are produced from the convolution of multiple wavelengths and modes. Two possible methods for analyzing this type of data are presented-one using Fourier transform deconvolution to extract resonance components from interference spectra, and another using chemometrics by constructing a partial least-squares model. Using isopropyl alcohol and water mixtures, detection limits are the order of 10-6 RIU (refractive index units), comparable to existing ring resonators devices. To study surface detectability and biomolecule detection feasibility, surface adsorption of bovine serum albumin (BSA) is also analyzed. [ABSTRACT FROM AUTHOR]

The proton affinity (PA) of 18-crown-6 (18C6) is determined using competitive threshold collision-induced dissociation (TCID) techniques. The PA of 18C6 is derived from four thermochemical cycles involving the relative thresholds for production of the protonated bases, H+(B), and protonated crown, H+(18C6), from the collision-induced dissociation (CID) of four proton bound heterodimers, (B)H+(18C6). The bases examined include glycine (Gly), alanine (Ala), imidazole (Imid), and 4-methylimidazole (4MeImid). In all cases, CID pathways for the loss of intact B and 18C6 are observed in competition. Loss of intact 18C6 is observed as the lowest-energy CID pathway for the (Imid)H+(18C6) and (4MeImid)H+(18C6) complexes. In contrast, loss of intact Gly and Ala is observed as the lowest-energy CID pathway for the (Gly)H+(18C6) and (Ala)H+(18C6) complexes, respectively. Excellent agreement between the measured and calculated (B)H+-18C6 and (18C6)H+-B bond dissociation energies (BDEs) is found with M06 theory, whereas B3LYP theory systematically underestimates these BDEs. On the basis of the relative TCID thresholds for the primary and competitive CID pathways, as well as the literature PAs of the bases, the PA of 18C6 is evaluated. The PA determined here for 18C6 exhibits excellent agreement with M06 and B3LYP theories, and very good agreement with the value reported by Meot-Ner determined using high pressure mass spectrometry (HPMS) techniques, suggesting that the PA of 18C6 reported in the NIST Webbook based on HPMS measurements by Kebarle and co-workers is overestimated. [ABSTRACT FROM AUTHOR]

Although qualitative and/or semiquantitative real-time monitoring of chemical reactions have been reported with a few mass spectrometric approaches, to our knowledge, no quantitative mass spectrometric approach has been reported so far to have a calibration valid up to molar concentrations as required by process control. This is mostly due to the absence of a practical solution that could well address the sample overloading issue. In this study, a novel autosampling flow injection analysis coupled with an atmospheric pressure chemical ionization mass spectrometry (FIA/APCI-MS) system, consisting of a 1 μL automatic internal sample injector, a postinjection splitter with 1:10 splitting ratio, and a detached APCI source connected to the mass spectrometer using a 4.5 in. long, 0.042 in. inner diameter (ID) stainless-steel capillary, was thus introduced. Using this system together with an optional FIA solvent modifier, e.g., 0.05% (v/v) isopropylamine, a linear quantitative calibration up to molar concentration has been achieved with 3.4-7.2% relative standard deviations (RSDs) for 4 replicates. As a result, quantitative real-time monitoring of a model reaction was successfully performed at the 1.63 M level. It is expected that this novel autosampling FIA/APCI-MS system can be used in quantitative real-time monitoring of a wide range of reactions under diverse reaction conditions. [ABSTRACT FROM AUTHOR]

Heparan sulfate (HS) glycosaminoglycans (GAGs) regulate a host of biological functions. To better understand their biological roles, it is necessary to gain understanding about the structure of HS, which requires identification of the sulfation pattern as well as the uronic acid epimerization. In order to model HS structure, it is necessary to quantitatively profile depolymerization products. To date, liquid chromatography-mass spectrometry (LC-MS) methods for profiling heparin lyase decomposition products have been shown. These enzymes, however, destroy information about uronic acid epimerization. Deaminative cleavage using nitrous acid (HONO) is a classic method for GAG depolymerization that retains uronic acid epimerization. Several chromatographic methods have been used for analysis of deaminative cleavage products. The chromatographic methods have the disadvantage that there is no direct readout on the structures producing the observed peaks. This report demonstrates a porous graphitized carbon (PGC)-MS method for the quantification of HONO generated disaccharides to obtain information about the sulfation pattern and uronic acid epimerization. Here, we demonstrate the separation and identification of uronic acid epimers as well as geometric sulfation isomers. The results are comparable to those expected for benchmark HS and heparin samples. The data demonstrate the utility of PGC-MS for quantification of HS nitrous acid depolymerization products for structural analysis of HS and heparin. [ABSTRACT FROM AUTHOR]

The presence of many salts, such as sodium chloride, can adversely affect the performance of native electrospray ionization mass spectrometry for the analysis of proteins and protein complexes by reducing the overall molecular ion abundances and distributing signal for any given charge state into many cationized forms with various numbers of adducts attached. Several solution additives, such as ammonium bromide, ammonium iodide, and NaSbF6, can significantly lower the extent of sodium ion adduction to the molecular ions of proteins and protein complexes. For ubiquitin, addition of 25 mM ammonium bromide or ammonium iodide into aqueous solutions also containing 1.0 mM NaCl results in a factor of 72 and 56 increase, respectively, in the relative abundances of the fully protonated molecular ions compared to when these additives are not present. The effectiveness of this method for reducing sodium ion adduction is related to the low proton affinity (PA) values of the anions. Anions with very low PA also have a propensity to adduct as an acid molecule, but these adducts can be readily dissociated from the molecular ions either by activation in the source or subsequently by collisional activation in the mass spectrometer. This method of reducing sodium ion adduction to proteins is simple and requires no experimental modifications, making it an attractive alternative to other methods for desalting proteins prior to mass spectrometry analysis. [ABSTRACT FROM AUTHOR]

Contemporary lipidomics protocols are dependent on conventional tandem mass spectrometry for lipid identification. This approach is extremely powerful for determining lipid class and identifying the number of carbons and the degree of unsaturation of any acyl-chain substituents. Such analyses are however, blind to isomeric variants arising from different carbon-carbon bonding motifs within these chains including double bond position, chain branching, and cyclic structures. This limitation arises from the fact that conventional, low energy collision-induced dissociation of even-electron lipid ions does not give rise to product ions from intrachain fragmentation of the fatty acyl moieties. To overcome this limitation, we have applied radical-directed dissociation (RDD) to the study of lipids for the first time. In this approach, bifunctional molecules that contain a photocaged radical initiator and a lipid-adducting group, such as 4-iodoaniline and 4-iodobenzoic acid, are used to form noncovalent complexes (i.e., adduct ions) with a lipid during electrospray ionization. Laser irradiation of these complexes at UV wavelengths (266 nm) cleaves the carbon-iodine bond to liberate a highly reactive phenyl radical. Subsequent activation of the nascent radical ions results in RDD with significant intrachain fragmentation of acyl moieties. This approach provides diagnostic fragments that are associated with the double bond position and the positions of chain-branching in glycerophospholipids, sphingomyelins and triacylglycerols and thus can be used to differentiate isomeric lipids differing only in such motifs. RDD is demonstrated for well-defined lipid standards and also reveals lipid structural diversity in olive oil and human very-low density lipoprotein. [ABSTRACT FROM AUTHOR]

Quantitative mass spectrometry methods offer near-comprehensive proteome coverage; however, these methods still suffer with regards to sample throughput. Multiplex quantitation via isobaric chemical tags (e.g., TMT and iTRAQ) provides an avenue for mass spectrometry-based proteome quantitation experiments to move away from simple binary comparisons and toward greater parallelization. Herein, we demonstrate a straightforward method for immediately expanding the throughput of the TMT isobaric reagents from 6-plex to 8-plex. This method is based upon our ability to resolve the isotopic shift that results from substituting a 15N for a 13C. In an accommodation to the preferred fragmentation pathways of ETD, the TMT-127 and -129 reagents were recently modified such that a 13C was exchanged for a 15N. As a result of this substitution, the new TMT reporter ions are 6.32 mDa lighter. Even though the mass difference between these reporter ion isotopologues is incredibly small, modern high-resolution and mass accuracy analyzers can resolve these ions. On the basis of our ability to resolve and accurately measure the relative intensity of these isobaric reporter ions, we demonstrate that we are able to quantify across eight samples simultaneously by combining the 13C- and 15N-containing reporter ions. Considering the structure of the TMT reporter ion, we believe this work serves as a blueprint for expanding the multiplexing capacity of the TMT reagents to at least 10-plex and possibly up to 18-plex. [ABSTRACT FROM AUTHOR]

Techniques for deriving confidence metrics for the reliability of automatically assigned elemental formulas in complex spectra, from high-resolution mass spectrometers, are described. These metrics can help an analyst to place an appropriate degree of trust in the results obtained from automated spectral analysis of, for example, natural organic materials. To provide these metrics of confidence, common mass spectrometric tests for reliability of peak assignment (mass accuracy/error, relative ion abundance, and rings-plus-double-bonds equivalence) are combined with novel confidence metrics based on the interconnectivity and consistency of a mass difference or mass defect based peak inference network and on the confidence of the initial library matches. These are shown to provide improved peak assignment confidence over manual or simple automatic assignment methods. [ABSTRACT FROM AUTHOR]