Cavities on a proteins surface as well as specific amino acid positioning within it create the physicochemical properties needed for a protein to perform its function. CASTp () is an online tool that locates and measures pockets and voids on 3D protein structures. This new version of CASTp includes annotated functional information of specific residues on the protein structure. The annotations are derived from the Protein Data Bank (PDB), Swiss-Prot, as well as Online Mendelian Inheritance in Man (OMIM), the latter contains information on the variant single nucleotide polymorphisms (SNPs) that are known to cause disease. These annotated residues are mapped to surface pockets, interior voids or other regions of the PDB structures. We use a semi-global pair-wise sequence alignment method to obtain sequence mapping between entries in Swiss-Prot, OMIM and entries in PDB. The updated CASTp web server can be used to study surface features, functional regions and specific roles of key residues of proteins.

A recent innovation in mass spectrometry is the ability to record mass spectra on ordinary samples, in their native environment, without sample preparation or preseparation by creating ions outside the instrument. In desorption electrospray ionization (DESI), the principal method described here, electrically charged droplets are directed at the ambient object of interest; they release ions from the surface, which are then vacuumed through the air into a conventional mass spectrometer. Extremely rapid analysis is coupled with high sensitivity and high chemical specificity. These characteristics are advantageously applied to high-throughput metabolomics, explosives detection, natural products discovery, and biological tissue imaging, among other applications. Future possible uses of DESI for in vivo clinical analysis and its adaptation to portable mass spectrometers are described.

A low-temperature plasma (LTP) probe has been developed for ambient desorption ionization. An ac electric field is used to induce a dielectric barrier discharge through use of a specially designed electrode configuration. The low-temperature plasma is extracted from the probe where it interacts directly with the sample being analyzed, desorbing and ionizing surface molecules in the ambient environment. This allows experiments to be performed without damage to the sample or underlying substrate and, in the case of biological analysis on skin surfaces, without electrical shock or perceptible heating. Positive or negative ions are produced from a wide range of chemical compounds in the pure stateand as mixtures in the gaseous, solution, or condensed phases, using He, Ar, N2, or ambient air as the discharge gas. Limited fragmentation occurs, although it is greater in the cases of the molecular than the atomic discharge gases. The effectiveness of the LTP probe has been demonstrated by recording characteristic mass spectra and tandem mass spectra of samples containing hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT) from poly(tetrafluoroethylene) (PTFE) surfaces where limits of detection are as low as 5 pg. Other performance characteristics, when using a commercial ion trap mass spectrometer, include 3-4 orders of magnitude linear dynamic range in favorable cases. Demonstration applications include direct analysis of cocaine from human skin, determination of active ingredients directly in drug tablets, and analysis of toxic and therapeutic compounds in complex biological samples. Ionization of chemicals directly from bulk aqueous solution has been demonstrated, where limits of detection are as low as 1 ppb. Large surface area sampling and control of fragmentation by a simple adjustment of the electrode configuration during operation are other demonstrated characteristics of the method.

Paper spray is developed as a direct sampling ionization method for mass spectrometric analysis of complex mixtures. Ions of analyte are generated by applying a high voltage to a paper triangle wetted with a small volume (<10 microL) of solution. Samples can be preloaded onto the paper, added with the wetting solution, or transferred from surfaces using the paper as a wipe. It is demonstrated that paper spray is applicable to the analysis of a wide variety of compounds, including small organic compounds, peptides, and proteins. Procedures are developed for analysis of dried biofluid spots and applied to therapeutic drug monitoring with whole blood samples and to illicit drug detection in raw urine samples. Limits of detection of 50 ng/mL (or 20 pg absolute) are achieved for atenolol in bovine blood. The combination of sample collection from surfaces and paper spray ionization also enables fast chemical screening at high sensitivity, for example 100 pg of heroin distributed on a surface and agrochemicals on fruit peels are detectable. Online derivatization with a preloaded reagent is demonstrated for analysis of cholesterol in human serum. The combination of paper spray with miniature mass spectrometers offers a powerful impetus to wide application of mass spectrometry in nonlaboratory environments.

The field of lipidomics has been significantly advanced by mass spectrometric analysis. The distinction and quantitation of the unsaturated lipid isomers, however, remain a long-standing challenge. In this study, we have developed an analytical tool for both identification and quantitation of lipid C=C location isomers from complex mixtures using online Paternò-Büchi reaction coupled with tandem mass spectrometry (MS/MS). The potential of this method has been demonstrated with an implementation into shotgun lipid analysis of animal tissues. Among 96 of the unsaturated fatty acids and glycerophospholipids identified from rat brain tissue, 50% of them were found as mixtures of C=C location isomers; for the first time, to our knowledge, the quantitative information of lipid C=C isomers from a broad range of classes was obtained. This method also enabled facile cross-tissue examinations, which revealed significant changes in C=C location isomer compositions of a series of fatty acids and glycerophospholipid (GP) species between the normal and cancerous tissues.Paternò-Büchi reaction | glycerophospholipids | photochemical reaction | lipid biomarkers | cancerous tissue analysis L ipids play a multitude of crucial roles in biological systems by serving as building blocks of cell membranes, sources for energy storage, and media for signal transduction (1-3). Unveiling the mechanisms and networks behind lipid homeostasis calls for sensitive, quantitative, and molecularly specific lipid analysis (4). The recent advancement in mass spectrometry (MS) for bioanalysis has enabled the field of lipidomics (5, 6) by allowing global identification and quantitation of lipid species at high speed (7-9) and providing information of lipid-lipid (10, 11) and lipidprotein interactions (12, 13) at systems level. These capabilities further expedite research on lipid biomarker discovery and metabolite flux analysis (14-16). Among many analytical figures of merit, high molecular specificity is a distinct feature of the MSbased approaches. Rich structural information of lipids in complex biological samples can now be routinely obtained, including the classes of the lipids, fatty acyl/alkyl composition, and even the sn positions of the fatty acyl/alkyl chains (17-19). The locations of the carbon-carbon double bonds (C=C) in the lipids, however, have rarely been identified using commercial MS systems and therefore have been either assumed or not reported in a large body of literatures for lipid study (20).The MS/MS methods, especially those involving low-energy collision-induced dissociation (CID), have not been effective in locating C=C bond locations, which is due to the high bond dissociation energies associated with cleaving a C=C bond. Without characteristic fragment ions produced, the C=C locations cannot be determined using MS/MS. To tackle this problem, two MS approaches have been explored, each with successes achieved but also with limitations observed. The first one employs C=C specific chemical derivatizations before MS analysis. T...

Intact, multiply protonated proteins of particular mass and charge were selected from ionized protein mixtures and gently landed at different positions on a surface to form a microarray. An array of cytochrome c, lysozyme, insulin, and apomyoglobin was generated, and the deposited proteins showed electrospray ionization mass spectra that matched those of the authentic compounds. Deposited lysozyme and trypsin retained their biological activity. Multiply charged ions of protein kinase A catalytic subunit and hexokinase were also soft-landed into glycerol-based liquid surfaces. These soft-landed kinases phosphorylated LRRASLG oligopeptide and D-fructose, respectively.

Mass spectrometry allows rapid chemical analysis of untreated samples in the ambient environment. This is a result of recent rapid progress in ambient ionization techniques. The most widely studied of these new methods, desorption electrospray ionization (DESI), uses fast-moving solvent droplets to extract analytes from surfaces and propel the resulting secondary microdroplets towards the mass analyzer. This review of DESI and other ambient methods centers on the accompanying chemical processes. Manipulation of the chemistry accompanying ambient ionization can be used to optimize chemical analysis, including molecular imaging. Solvent effects, geometry effects, electrochemical processes and mechanisms are covered. Extensions of the methodology to solution-phase analysis, to stand-off detection and to therapeutic drug analysis using miniature mass spectrometers are also treated.

A mass analyzer based on a rectilinear geometry ion trap (RIT) has been built, and its performance has been characterized. Design concepts for this type of ion trap are delineated with emphasis on the effects of electrode geometry on the calculated electric field. The Mathieu stability region was mapped experimentally. The instrument can be operated using mass-selective instability scans in both the boundary and resonance ejection versions. Comparisons of performance between different versions of the device having different dimensions allowed selection of an optimized geometry with an appropriate distribution of higher-order electric fields. Comparisons made under the same conditions between the performance of a conventional cylindrical ion trap and a RIT of 4 times greater volume show an improvement of 40 times in the signal-to-noise ratio resulting from the higher ion trapping capacity of the RIT. The demonstrated capabilities of the RIT include tandem mass spectrometry, a mass resolution in excess of 1000, and a mass/charge range of 650 Th, all in a simple structure that is only 3.5 cm(3) in internal volume.