Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200-microm i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. With gradients of acetonitrile in 100 mM triethylammonium acetate, the synthesized columns allowed the rapid and highly efficient separation of single-stranded oligodeoxynucleotides and double-stranded DNA fragments by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). Compared with capillary columns packed with micropellicular, octadecylated poly-(styrene/divinylbenzene) particles, an improvement in column performance of approximately 40% was obtained, enabling the analysis of an 18-mer oligodeoxynucleotide with a column efficiency of more than 190000 plates per meter. The chromatographic separation system was on-line-coupled to electrospray ionization mass spectrometry (ESI-MS). To improve the mass spectrometric detectabilities, 25 mM triethylammonium bicarbonate was utilized as an ion-pair reagent at the cost of only little reduction in separation performance and acetonitrile was added postcolumn as the sheath liquid through the triaxial electrospray probe. High-quality mass spectra of femtomole amounts of 3-mer to 80-mer oligodeoxynucleotides were recorded showing very little cation adduction. Double-stranded DNA fragments ranging in size from 51 to 587 base pairs were separated and detected by IP-RP-HPLC-ESI-MS. Accurate mass determination by deconvolution of the mass spectra was feasible for DNA fragments up to the 267-mer with a molecular mass of 165 019, whereas the spectra of longer fragments were too complex for deconvolution because of incomplete separation due to overloading of the column. Finally, on-line IP-RP-HPLC tandem MS was applied to the sequencing of short oligodeoxynucleotides.

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.

A sophisticated matching algorithm developed for highly efficient identity search within tandem mass spectral libraries is presented. For the optimization of the search procedure a collection of 410 tandem mass spectra corresponding to 22 compounds was used. The spectra were acquired in three different laboratories on four different instruments. The following types of tandem mass spectrometric instruments were used: quadrupole-quadrupole-time-of-flight (QqTOF), quadrupole-quadrupole-linear ion trap (QqLIT), quadrupole-quadrupole-quadrupole (QqQ), and linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LIT-FTICR). The obtained spectra were matched to an established MS/MS-spectral library that contained 3759 MS/MS-spectra corresponding to 402 different reference compounds. All 22 test compounds were part of the library. A dynamic intensity cut-off, the search for neutral losses, and optimization of the formula used to calculate the match probability were shown to significantly enhance the performance of the presented library search approach. With the aid of these features the average number of correct assignments was increased to 98%. For statistical evaluation of the match reliability the set of fragment ion spectra was extended with 300 spectra corresponding to 100 compounds not included in the reference library. Performance was checked with the aid of receiver operating characteristic (ROC) curves. Using the magnitude of the match probability as well as the precursor ion mass as benchmarks to rate the obtained top hit, overall correct classification of a compound being included or not included in the mass spectrometric library, was obtained in more than 95% of cases clearly indicating a high predictive accuracy of the established matching procedure.

The potential of the combined use of ESI-QqTOF-MS and ESI-QqTOF-MS/MS with mass-spectral library search for the identification of therapeutic and illicit drugs has been evaluated. Reserpine was used for standardizing experimental conditions and for characterization of the performance of the applied mass spectrometric system. Experiments revealed that because of the mass accuracy, the stability of calibration, and the reproducibility of fragmentation, the QqTOF mass spectrometer is an appropriate platform for establishment of a tandem-mass-spectral library. Three-hundred and nineteen substances were used as reference samples to build the spectral library. For each reference compound, product-ion spectra were acquired at ten different collision-energy values between 5 eV and 50 eV. For identification of unknown compounds, a library search algorithm was developed. The closeness of matching between a measured product-ion spectrum and a spectrum stored in the library was characterized by a value called "match probability", which took into account the number of matched fragment ions, the number of fragment ions observed in the two spectra, and the sum of the intensity differences calculated for matching fragments. A large value for the match probability indicated a close match between the measured and the reference spectrum. A unique feature of the library search algorithm-an implemented spectral purification option-enables characterization of multi-contributor fragment-ion spectra. With the aid of this software feature, substances comprising only 1.0% of the total amount of binary mixtures were unequivocally assigned, in addition to the isobaric main contributors. The spectral library was successfully applied to the characterization of 39 forensic casework samples.

The numerous problems posed by modern biochemistry, biology, and medicine, as well as the growing significance of genetic engineering require the application of fast and reliable methods of utmost sensitivity and selectivity for the analysis of nucleic acids. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) represent established analytical techniques for the characterization and structural elucidation of single- and double-stranded nucleic acids, ranging in size from a few nucleotides to several thousand base pairs. Although both techniques are independently applicable for nucleic acid analysis, the on-line hyphenation significantly enhances their potential for the robust and fully automable routine analysis of minute amounts of biological samples. Among the various chromatographic and mass spectrometric modes available in principle, ion-pair reversed-phase HPLC and electrospray ionization mass spectrometry (ESI-MS) have been shown to be the most suitable for the direct interfacing of liquid chromatography (LC) and MS. Instrumental setup, as well as chromatographic and mass spectrometric experimental conditions, need to be carefully selected in order to maximize the performance of the hyphenated analytical system. Applications of HPLC-ESI-MS include the characterization of oligodeoxynucleotides synthesized by solid-phase synthesis, the analysis of antisense oligodeoxynucleotides, oligonucleotide metabolites, and DNA adducts, the analysis of genomic segments specifically amplified by the polymerase chain reaction (PCR), the characterization of ribonucleic acids, the sizing of double-stranded DNA restriction fragments, the genotyping of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), the detection of mutations in nucleic acid sequences, and the sequencing of nucleic acids.

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The inter-instrument and inter-laboratory transferability of a tandem mass spectral reference library originally built on a quadrupole-quadrupole-time-of-flight instrument was examined. The library consisted of 3759 MS/MS spectra collected from 402 reference compounds applying several different collision-energy values for fragmentation. In the course of the multicenter study, 22 test compounds were sent to three different laboratories, where 418 tandem mass spectra were acquired using four different instruments from two manufacturers. The study covered the following types of tandem mass spectrometers: quadrupole-quadrupole-time-of-flight, quadrupole-quadrupole-linear ion trap, quadrupole-quadrupole-quadrupole, and linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. In each participating laboratory, optimized instrumental parameters were gathered solely from routinely applied workflows. No standardization procedure was applied to increase the inter-instrument comparability of MS/MS spectra. The acquired tandem mass spectra were matched against the established reference library using a sophisticated matching algorithm, which is presented in detail in a companion paper. Correct answers, meaning that the correct compound was retrieved as top hit, were obtained in 98.1% of cases. For the remaining 1.9% of spectra, the correct compound was matched at second rank. The observed high percentage of correct assignments clearly suggests that the developed mass spectral library search approach is to a large extent platform independent.

ObjectiveNicotinamide phosphoribosyltransferase (NAMPT, also referred to as pre-B cell colony-enhancing factor or visfatin) is critically required for the maintenance of cellular nicotinamide adenine dinucleotide (NAD) supply catalysing the rate-limiting step of the NAD salvage pathway. NAMPT is strongly upregulated in inflammation including IBD and counteracts an increased cellular NAD turnover mediated by NAD-depleting enzymes. These constitute an important mechanistic link between inflammatory, metabolic and transcriptional pathways and NAD metabolism.DesignWe investigated the impact of NAMPT inhibition by the small-molecule inhibitor FK866 in the dextran sulfate sodium (DSS) model of colitis and the azoxymethane/DSS model of colitis-associated cancer. The impact of NAD depletion on differentiation of mouse and human primary monocytes/macrophages was studied in vitro. Finally, we tested the efficacy of FK866 compared with dexamethasone and infliximab in lamina propria mononuclear cells (LPMNC) isolated from patients with IBD.ResultsFK866 ameliorated DSS-induced colitis and suppressed inflammation-associated tumorigenesis in mice. FK866 potently inhibited NAMPT activity as demonstrated by reduced mucosal NAD, resulting in reduced abundances and activities of NAD-dependent enzymes including PARP1, Sirt6 and CD38, reduced nuclear factor kappa B activation, and decreased cellular infiltration by inflammatory monocytes, macrophages and activated T cells. Remarkably, FK866 effectively supressed cytokine release from LPMNCs of patients with IBD. As FK866 was also effective in Rag1−⁄− mice, we mechanistically linked FK866 treatment with altered monocyte/macrophage biology and skewed macrophage polarisation by reducing CD86, CD38, MHC-II and interleukin (IL)-6 and promoting CD206, Egr2 and IL-10.ConclusionOur data emphasise the importance of NAD immunometabolism for mucosal immunity and highlight FK866-mediated NAMPT blockade as a promising therapeutic approach in acute intestinal inflammation.