Small Molecule Mass Research Articles

Conjugated Three-Dimensional High-Connected Covalent Organic Frameworks for Lithium-Sulfur Batteries.

Developing conjugated three-dimensional (3D) covalent organic frameworks (COFs) still remains an extremely difficult task due to the lack of enough conjugated 3D building blocks. Herein, condensation between an 8-connected pentiptycene-based D2h building block (DMOPTP) and 4-connected square-planar linkers affords two 3D COFs (named 3D-scu-COF-1 and 3D-scu-COF-2). A combination of the 3D homoaromatic conjugated structure of the former building block with the 2D conjugated structure of the latter linking units enables the π-electron delocalization over the whole frameworks of both COFs, endowing them with excellent conductivities of 3.2-3.5 × 10-5 S cm-1. In particular, the 3D rigid quadrangular prism shape of DMOPTP guides the formation of a twofold interpenetrated scu 3D topology and high-connected permanent porosity with a large Brunauer-Emmett-Teller (BET) surface area of 2340 and 1602 m2 g-1 for 3D-scu-COF-1 and 3D-scu-COF-2, respectively, ensuring effective small molecule storage and mass transport characteristics. This, in combination with their good charge transport properties, renders them promising sulfur host materials for lithium-sulfur batteries (LSBs) with high capacities (1035-1155 mA h g-1 at 0.2 C, 1 C = 1675 mA g-1), excellent rate capabilities (713-757 mA h g-1 at 5.0 C), and superior cycling stability (71-83% capacity retention at 2.0 C after 500 cycles), surpassing the most of organic LSB cathodes reported thus far.

Annotation of complex mass spectra by multi-layered analysis

Annotating electrospray small molecule mass spectra remains a challenging problem due to the multiple processes occurring during ionization. Although an [M+H]+ is often present, ions can be formed by reactions with other cations, background compounds and co-eluting species, and by in-source fragmentation. Even single analytes can produce multiple ion forms, many of which remain unidentified and may appear to be different species, affecting reproducibility, quantification and precursor selection in DDA experiments. Annotation usually compares differences between peaks to known adducts and losses but fails if key peaks are missing or if the peaks are from unexpected adducts. Further, isotopes are often assumed to be due to 13C and removed prior to analysis which can leave ‘orphan’ peaks if unusual elements are present. Here we describe an alternative multi-layered approach (MLA) which successively matches spectra to calculated target ion lists and reprocesses the residual ions. This allows the analyst to focus on the unknown ions and to progressively increase target list complexity since explained ions are removed. Target ion lists can be calculated from expected or observed masses and potential adducts or can be pre-defined lists, for example common contaminants. Using this approach on spectra of known standards we identified adducts with Ca, Al, Fe, Ba and possibly Mg and Sr. We also detected several compounds and adducts in a spectrum of co-eluting species from an LC-MS analysis.

Highly Multiplexed Immunohistochemical MALDI-MS Imaging of Biomarkers in Tissues.

Immunohistochemistry (IHC) combined with fluorescence microscopy provides an important and widely used tool for researchers and pathologists to image multiple biomarkers in tissue specimens. However, multiplex IHC using standard fluorescence microscopy is generally limited to 3–5 different biomarkers, with hyperspectral or multispectral methods limited to 8. We report the development of a new technology based on novel photocleavable mass-tags (PC-MTs) for facile antibody labeling, which enables highly multiplexed IHC based on MALDI mass spectrometric imaging (MALDI-IHC). This approach significantly exceeds the multiplexity of both fluorescence- and previous cleavable mass-tag-based methods. Up to 12-plex MALDI-IHC was demonstrated on mouse brain, human tonsil, and breast cancer tissues specimens, reflecting the known molecular composition, anatomy, and pathology of the targeted biomarkers. Novel dual-labeled fluorescent PC-MT antibodies and label-free small-molecule mass spectrometric imaging greatly extend the capability of this new approach. MALDI-IHC shows promise for use in the fields of tissue pathology, tissue diagnostics, therapeutics, and precision medicine.

Physical and chemical behaviour of tungsten oxide in the presence of nickel additive under hydrogen and carbon monoxide atmospheres

The physical and chemical behaviour of bulk tungsten oxide (WO3) and Ni doped tungsten oxide (15% Ni/WO3) were examined by performing a temperature-programmed reduction (TPR) technique. The chemical composition, morphology, and surface composition of both samples before and after reduced were analysed by X-ray diffraction (XRD), scanning electron microscopy (FESEM), and X-ray photoelectron spectroscopy (XPS) analysis. The XRD pattern of calcined Ni doped tungsten oxide powder comprised of WO3 and nickel tungstate (NiWO4) phases. The reduction behaviour was investigated by a non-isothermal reduction up to 900 °C achieved under (10 and 20% v/v) hydrogen in nitrogen (H2 in N2) and (20 and 40% v/v) carbon monoxide in nitrogen (CO in N2) atmospheres. The H2-TPR were indicated the reduction of bulk WO3 and 15% NiWO3 proceed in three steps (WO3 → WO2 → WO2 + W) and (WO3 → WO2 → W + Ni4W) respectively under 20% H2. Whereas, the reduction of 15% WO3 under 40% CO involves of two following stages: (i) low temperature (<800 °C) transformation of WO3 → WO2.72 → WO2 and, (ii) high temperature (>800 °C) transformation of WO2 → W → WC. Furthermore, NiWO4 alloy phase was transformed according to the sequence NiWO4 → Ni + WO2.72 → Ni + WO2 → Ni + W → Ni4W + W at temperature >700 °C and >800 °C in H2 and CO atmospheres, respectively. It can be concluded that the reduction behaviour of WO3 is matched with the thermodynamic data. In addition, the reduction under H2 is more favourable and have better reducibility compared to the CO gas. It is due to the small molecule size and molecule mass of H2 that encourages the diffusion of H2 molecule into the internal surface of the catalyst compared to CO. Moreover, Ni additive had improved the WO3 reducibility and enhancing the CO adsorption and promotes the formation of tungsten carbide (WC) by carburisation reaction. Besides, the formation of Ni during the reduction of 15% Ni/WO3 under CO reductant catalysed the Boudouard reaction to occur, which disproportionated the carbon monoxide to carbon dioxide and carbon (CO → CO2 + C).

Effect of Solvent Quality on Laminar Slip Flow Penetration of Poly(N-isopropylacrylamide) Films with an Exploration of the Mass Transport Mechanism.

The effect of solvent quality on the slip flow penetration of polymer films was evaluated by monitoring small-molecule mass transport under varying laminar flow rates using Förster resonance energy transfer in combination with total internal reflectance fluorescence microscopy (FRET-TIRFM). For thin films of poly(N-isopropylacrylamide) (pNIPAM), solvents with solvent quality ranging from good to poor were studied. The solvents used were composed of varying mole ratios of methanol and water in order to take advantage of the unique cononsolvency phenomenon of pNIPAM such that differences in the physicochemical properties of these solvents were insignificant for fluorescence detection. FRET quenching of a donor fluorophore covalently tethered on the substrate surface at the bottom of the pNIPAM film by a solution-confined acceptor was monitored as a function of time. Quenching curves were fit to a combined Taylor-Aris-Fickian mass transport model for the acceptor, rhodamine B (RhB) or 2-nitrobenzylaclohol (2-NBA), allowing apparent diffusion coefficients to be determined and used to assess slip flow penetration into the polymer film. An increase in the apparent diffusion coefficient of tracer molecules was observed with increasing laminar flow rate for all solvents, indicating that mass transport processes in the pNIPAM film are significantly perturbed by laminar slip flow penetration. In going from poor solvents, 31 mol % MeOH/H2O and 20 mol % MeOH/H2O, to the theta solvent, 13 mol % MeOH/H2O, and finally to a good solvent, 100% methanol, the slip length increases from 25 to 37 to 70 to 128 nm, with the corresponding percentage of the film penetrated by slip flow increasing from 19 to 27 to 42 to 57%, respectively. The apparent diffusion coefficients of the two acceptors, RhB and 2-NBA, which differ substantially in size and charge, in pNIPAM films under identical conditions were found to be of the same order of magnitude, albeit with a small difference (∼10%) due to inherently different diffusive properties. Therefore, the dominant mechanism for the mass transport of small molecules in densely grafted thin pNIPAM brush films is suggested to be linear Fickian diffusion under the chosen laminar flow conditions with linear flow velocities ranging from 192 to 2952 μm/s. High-quality fits to a Taylor-Aris-Fickian diffusion model of the experimental breakthrough curves obtained with both acceptor molecules further substantiate the proper use of this model and the validity of the FRET-TIRFM method.

Optimizing cell-free protein expression in CHO: Assessing small molecule mass transfer effects in various reactor configurations.

Cell-free protein synthesis (CFPS) is an ideal platform for rapid and convenient protein production. However, bioreactor design remains a critical consideration in optimizing protein expression. Using turbo green fluorescent protein (tGFP) as a model, we tracked small molecule components in a Chinese Hamster Ovary (CHO) CFPS system to optimize protein production. Here, three bioreactors in continuous-exchange cell-free (CECF) format were characterized. A GFP optical sensor was built to monitor the product in real-time. Mass transfer of important substrate and by-product components such as nucleoside triphosphates (NTPs), creatine, and inorganic phosphate (Pi) across a 10-kDa MWCO cellulose membrane was calculated. The highest efficiency measured by tGFP yields were found in a microdialysis device configuration; while a negative effect on yield was observed due to limited mass transfer of NTPs in a dialysis cup configuration. In 24-well plate high-throughput CECF format, addition of up to 40 mM creatine phosphate in the system increased yields by up to ∼60% relative to controls. Direct ATP addition, as opposed to creatine phosphate addition, negatively affected the expression. Pi addition of up to 30 mM to the expression significantly reduced yields by over ∼40% relative to controls. Overall, data presented in this report serves as a valuable reference to optimize the CHO CFPS system for next-generation bioprocessing. Biotechnol. Bioeng. 2017;114: 1478-1486. © 2017 Wiley Periodicals, Inc.

Oral transfer of chemical cues, growth proteins and hormones in social insects.

Social insects frequently engage in oral fluid exchange - trophallaxis - between adults, and between adults and larvae. Although trophallaxis is widely considered a food-sharing mechanism, we hypothesized that endogenous components of this fluid might underlie a novel means of chemical communication between colony members. Through protein and small-molecule mass spectrometry and RNA sequencing, we found that trophallactic fluid in the ant Camponotus floridanus contains a set of specific digestion- and non-digestion related proteins, as well as hydrocarbons, microRNAs, and a key developmental regulator, juvenile hormone. When C. floridanus workers' food was supplemented with this hormone, the larvae they reared via trophallaxis were twice as likely to complete metamorphosis and became larger workers. Comparison of trophallactic fluid proteins across social insect species revealed that many are regulators of growth, development and behavioral maturation. These results suggest that trophallaxis plays previously unsuspected roles in communication and enables communal control of colony phenotypes.

Self-reporting hybridisation assay for miRNA analysis

Hybridisation assays, which are commonly used to analyse oligonucleotides such as siRNAs and miRNAs, often employ detection probes with fluorescent tags. The signal emitted by a fluorescent tag covers a broad range of wavelengths and this limits the multiplexing potential due to overlapping signals. A novel method of indirect oligonucleotide analysis has been developed which combines a hybridisation assay with cleavable small molecule mass tags using HPLC-ESI MS detection. A self-reporting detection probe has been designed which incorporates a DNA/RNA chimeric oligonucleotide sequence in the reporter region, which generates small nucleotide products upon RNase cleavage of the ribose-phosphate backbone. These small nucleotides can then serve as mass tags for the indirect detection of oligonucleotide analytes. The narrow mass range covered by a small molecule mass tag combined with the wide range of possible mass tags provides a high degree of multiplexing potential. This approach has been demonstrated for the analysis of a synthetic miRNA.

A Genetic Basis for Donor Variation in Generation of Prostaglandins and Leukotrienes in Stored RBCs Using a Mouse Model

Abstract 844 Background:RBC transfusion has clear efficacy in treating various types of anemia. However, in recent decades, there is a renewed focus on the potential for negative clinical sequelae from transfusing stored RBCs. Whether or not older, stored RBC units are associated with adverse outcomes remains controversial. However, there are clear cellular and biochemical data that RBC units accumulate particles and molecules over time with known toxicity when administered to animals and/or humans. Among the most potent of these are prostaglandins and leukotrienes (jointly known as eicosanoids), which are potent mediators of inflammation and vascular pathology. Indeed, arachidonic acid (AA), and 5-, 12-, and 15-hydroxyeicsotetranoic acid (HETE) accumulate during storage of human RBCs and are biologically active in priming neutrophils (Silliman et al., Transfusion 2011 51(12):2549-54). It is well known that there is substantial donor-to-donor variation in how well RBCs store from the standpoint of post-transfusion RBC recovery; however, it is unclear whether the accumulation of eicosanoids varies substantially among donors. If differences exist, then it may be useful to screen RBC units prior to transfusion into patients with illnesses likely to be affected by eicosanoid exposure. Using a well characterized mouse model of RBC storage, and different strains of donor mice, we tested the hypothesis that there are genetic determinants affecting eicosanoid levels in stored RBCs. Methods:RBCs from C57BL/6 (B6) and FVB mice were collected in CPDA-1, filter leukoreduced, and stored under conditions previously shown to model human RBC storage. Samples collected on days 0, 5, 9, and 14 were analyzed by small molecule mass spectrometry. The study was repeated 3 times and combined data were analyzed. Results:AA accumulated over storage time in both B6 and FVB RBC units to a similar level. In contrast, although essentially no accumulation of eicosanoids was observed in B6 RBC units, substantial time-dependent increases (compared to day of collection) were observed in FVB RBC units for 5-HETE (4-fold) and 15-HETE (12-fold). In addition, a greater than 10-fold increase was observed for prostaglandin E2 in FVB RBC units with no detectable prostaglandin E2 in B6 RBC units [ findings were consistent in all 3 experiments and all differences had p values of <.05]. As oxidized phospholipids are better substrates for phospholipases that generate AA, we hypothesized that oxidative damage would be increased in FVB RBCs. Additional metabolomic analysis demonstrated that B6 RBCs had decreased levels of oxidative damage compared to FVB RBCs, including substantially lower levels of 9,10-epoxystearate, an oxidized membrane lipid. Consumption of glucose and production of lactate were similar in both strains; however, B6 RBCs had increased total glutathione (GSH), oxidized GSH, and cysteine-GSH disulfides. No difference in flux through the pentose-phosphate shunt was observed (including similar NADPH levels); this suggests that the increased GSH in C57BL/6 RBCs was due to de novo synthesis and not to regeneration by GSH reductase. C57BL/6 RBCs also had increased levels of natural anti-oxidants, including ergothioneine and alpha-tocopherol. [All the above differences achieved a p value of <.05]. Conclusion:The current findings take advantage of a mouse model of RBC storage. Given that two inbred, genetically distinct strains demonstrate significant differences in the rate and magnitude of eicosanoid generation, these findings strongly support both the existence of donor variability in eicosanoid accumulation during RBC storage and a genetic basis thereof (in the context of a mouse system). In addition to providing a tractable platform to dissect the genetics of donor-specific RBC storage biology, these findings suggest the hypothesis that similar differences in eicosanoid generation occur with human RBC storage. Subsequent analysis of human specimens will need to be performed to test whether these findings extend to stored human RBC units. Disclosures:No relevant conflicts of interest to declare.

Use of an Artificial Immune System Derived Method for the Charge State Assignment of Small-Molecule Mass Spectra

Knowing the charge state of an ion in a mass spectrum is crucial to being able to assign a formula to it. For many small-molecule peaks in complex mass spectra, the intensities of the isotopic peaks are too low to allow the charge state to be calculated from isotopic spacings, which is the basis of the conventional method of determining the charge state of an ion. A novel artificial intelligence derived method for identifying the charge state of ions, in the absence of any isotopic information or a series of charge states, has been developed using an artificial immune system approach. This technique has been tested against synthetic and real data sets and has proven successful in identifying the majority of multiply charged ions, thereby significantly improving the peak assignment rate and confidence.

An assessment of current bioinformatic solutions for analyzing LC-MS data acquired by selected reaction monitoring technology.

Selected reaction monitoring (SRM) is an accurate quantitative technique, typically used for small-molecule mass spectrometry (MS). SRM has emerged as an important technique for targeted and hypothesis-driven proteomic research, and is becoming the reference method for protein quantification in complex biological samples. SRM offers high selectivity, a lower limit of detection and improved reproducibility, compared to conventional shot-gun-based tandem MS (LC-MS/MS) methods. Unlike LC-MS/MS, which requires computationally intensive informatic postanalysis, SRM requires preacquisition bioinformatic analysis to determine proteotypic peptides and optimal transitions to uniquely identify and to accurately quantitate proteins of interest. Extensive arrays of bioinformatics software tools, both web-based and stand-alone, have been published to assist researchers to determine optimal peptides and transition sets. The transitions are oftentimes selected based on preferred precursor charge state, peptide molecular weight, hydrophobicity, fragmentation pattern at a given collision energy (CE), and instrumentation chosen. Validation of the selected transitions for each peptide is critical since peptide performance varies depending on the mass spectrometer used. In this review, we provide an overview of open source and commercial bioinformatic tools for analyzing LC-MS data acquired by SRM.

Self reporting RNA probes as an alternative to cleavable small molecule mass tags

The large size of biological molecules such as proteins and oligonucleotides makes them inherently problematic to analyse and quantify directly by mass spectrometry. For these molecules, electrospray ionisation produces multiply charged species and associated alkali metal adducts which can reduce sensitivity and complicate quantification. Whereas time-of-flight mass analysers, often coupled to matrix-assisted laser desorption/ionisation, can have insufficient mass resolution to resolve these large molecules in the higher m/z range. This has led to the development of cleavable small molecule mass tag approaches for the indirect analysis of biomolecules such as proteins and oligonucleotides. Existing methodologies require the design and synthesis of a cleavable linker to join the biomolecule and the mass tag. Here, an alternative approach to small molecule mass tags is presented, which exploits the properties of the RNA molecule to afford self-reporting probes which can be easily synthesised using automated phosphoramidite chemistry. The sugar-phosphate backbone of RNA was used as a built-in enzyme cleavable linker and through the use of RNase digestion of bromine labelled oligonucleotides the observation of a range of small molecule mass tags by mass spectrometry is demonstrated. This study provides a proof-of-concept that RNase digestion can be used to produce labelled small molecule mass tags from oligonucleotide probes, thus eliminating the need for custom design and synthesis of a cleavable linker.

Small-molecule analysis by surface-assisted laser desorption/ionization mass spectrometry

In order to meet the challenges facing modern chemistry, biology, and medicine, methods are required capable of performing rapid and reliable analysis of both individual compounds and complex mixtures at the molecular level. Matrix-assisted laser de-sorption/ionization mass spectrometry meets these requirements; however, some limitations complicate its application for the analysis of small molecules. Recently, small-molecule analysis has greatly progressed owing to development of surface-assisted laser desorption/ionization mass spectrometry involving approaches which combine the unique properties of nanostructured surface chemistry and morphology. This review examines such approaches and their specific application in small-molecule mass analysis.

The cAMP Capture Compound Mass Spectrometry as a Novel Tool for Targeting cAMP-binding Proteins : FROM PROTEIN KINASE A TO POTASSIUM/SODIUM HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATED CHANNELS

The profiling of subproteomes from complex mixtures on the basis of small molecule interactions shared by members of protein families or small molecule interaction domains present in a subset of proteins is an increasingly important approach in functional proteomics. Capture CompoundTM Mass Spectrometry (CCMS) is a novel technology to address this issue. CCs are trifunctional molecules that accomplish the reversible binding of target protein families to a selectivity group (small molecule), covalent capturing of the bound proteins by photoactivated cross-linking through a reactivity group, and pullout of the small molecule-protein complexes through a sorting function, e.g. biotin. Here we present the design, synthesis, and application of a new Capture Compound to target and identify cAMP-binding proteins in complex protein mixtures. Starting with modest amounts of total protein mixture (65–500 μg), we demonstrate that the cAMP-CCs can be used to isolate bona fide cAMP-binding proteins from lysates of Escherichia coli, mammalian HepG2 cells, and subcellular fractions of mammalian brain, respectively. The identified proteins captured by the cAMP-CCs range from soluble cAMP-binding proteins, such as the catabolite gene activator protein from E. coli and regulatory subunits of protein kinase A from mammalian systems, to cAMP-activated potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channels from neuronal membranes and specifically synaptosomal fractions from rat brain. The latter group of proteins has never been identified before in any small molecule protein interaction and mass spectrometry-based proteomics study. Given the modest amount of protein input required, we expect that CCMS using the cAMP-CCs provides a unique tool for profiling cAMP-binding proteins from proteome samples of limited abundance, such as tissue biopsies.

Single-Cell Transfection by Electroporation Using an Electrolyte/Plasmid-Filled Capillary

Single-cell transfection of adherent cells has been accomplished using single-cell electroporation (SCEP) with a pulled capillary. HEPES-buffered physiological saline solution containing pEGFP plasmid at a low concentration (0.16 approximately 0.78 microg/microL) filled a 15 cm long capillary with a tip opening of 2 microm. The electric field is applied to individual cells by bringing the tip close to the cell and subsequently applying one or two brief electric pulses. Many individual cells can thus be transfected with a small volume of plasmid-containing solution (approximately 1 microL). The extent of electroporation is determined by measuring the percentage loss of freely diffusing thiols (chiefly reduced glutathione) that have been derivatized with the fluorogenic ThioGlo 1. A mass transport model is used to fit the time-dependent fluorescence intensity decay in the target cells. The fits, which are excellent, yield the electroporation-induced fluorescence loss at steady state and the mass transfer rate through the electroporated cell membrane. Steady-state fluorescence loss ranged approximately from 0 to about 80% (based on the fluorescence intensity before electroporation). For the cells having a loss of thiol-ThioGlo 1 fluorescence intensity greater than 10% and mass transfer rate greater than 0.03 s(-1), EGFP fluorescence is observed after 24 h. The EGFP fluorescence is increased at 48 h. With a loss smaller than 10% and a mass transfer rate smaller than 0.03 s(-1), no EGFP fluorescence is detected. Thus, transfection success is closely related to the small molecule mass transport dynamics as indicated by the loss of fluorescence from thiol-ThioGlo 1 conjugates. The EGFP expression is weaker than bulk lipid-mediated transfection, as indicated by the EGFP fluorescence intensities. However, the success with the single-cell approach is considerably greater than lipid-mediated transfection.

Mapping a plant's chemical vocabulary

Plants generate an amazing variety of small molecules and are arguably nature's finest chemists. A new study identifies over 2,000 small molecule mass peaks in the model plant Arabidopsis thaliana and defines both the genetic diversity and genetic architecture controlling the production of these compounds.

Structural Characterization of in Vitro and in Vivo Intermediates on the Loading Module of Microcystin Synthetase

The microcystin family of toxins is the most common cause of hepatotoxicity associated with water blooms of cyanobacterial genera. The biosynthetic assembly line producing the toxic cyclic peptide, microcystin, contains an adenylation-peptidyl carrier protein didomain (A-PCP) at the N-terminus of the initiator module McyG (295 kDa) that has been postulated to activate and load the starter unit phenylacetate for formation of the unusual aromatic beta-amino acid residue, Adda, before subsequent extension. Characterization of the McyG A-PCP didomain (78 kDa) using ATP-PP i exchange assays and mass spectrometry revealed that assorted phenylpropanoids are preferentially activated and loaded onto the PCP carrier domain rather than phenylacetate itself. For the first time, thioesters formed in vivo were detected directly using large molecule mass spectrometry. Additionally substrates were cleaved using a type II thioesterase for structural elucidation by small molecule mass spectrometry. Unprecedented features of the McyG A-PCP didomain include the in vivo acylation of the holo PCP with exogenous and endogenous substrates, along with the ability of the apo protein to retain the acyl-AMP intermediate during affinity purification. These results imply that phenylpropanoids are preferentially loaded onto the McyG PCP; however one carbon must be excised following extension of the starter unit with malonyl-CoA in order to generate the expected polyketide chain which leads us to ponder the novel biochemistry by which this occurs.

Dynamic lipidomic insights into phosphatidylcholine synthesis from organelle to organism

Recent technical improvement and technological innovation in small molecule mass spectrometry have provided powerful tools for the intensive metabolomic biochemical investigations that will necessarily characterise the ”post‐genomic“ era of biomedical research. For membrane phospholipids, use of tandem electrospray ionisation mass spectrometry (ESI‐MS/MS) exploiting precursor scanning of class‐specific diagnostic fragments, can provide detailed quantitative profiles at the level of individual molecular species for many hundreds of unique lipids. Such ”snapshot“ measurements provide little information concerning metabolic flux. However, recent use of metabolic labelling with stable isotope derivatives of phospholipid headgroups combined with precursor scans of unlabelled and labelled fragments have yielded considerable insight into phosphatidylcholine metabolism in vivo. Here, we briefly review some of the recent work on pathways of phosphatidylcholine metabolism ranging from studies at subcellular organelle level through to whole organism. The sensitivity, specificity and suitability of this powerful methodological approach to numerous questions of phospholipid metabolism place ESI‐MS/MS at the very heart of dynamic lipidomics in the foreseeable future.

Short Daily Hemodialysis vs. Short Daily Hemofiltration (Search for Optimal Prescription)

It has been shown that daily hemodialysis as well as convective transfer by hemofilitration improve the quality of extra renal treatment. Two following phases of treatment of three weeks each were tested in 2 patients: daily hemodialysis 2.5 h 6 times/week (HD*6) and daily hemofiltration 2.75 h 6 times/week (HF*6) performed according to the following modalities. Phase I, blood flow rates (QB): 300 mL/min, hemofilter 1.4 m2AN 69 dialysate flow 500 mL/min. Phase II, QB: 150 mL/min, hemofilter 1m2AN 69, exchange volume of 10 L/session; 5 L predilution and 5 L postdilution (conditions were limited by the device). We measured, during the third week of treatment of each phase, the weekly mass transfers and the predialysis plasma levels of urea (U), creatinine (C), phosphate (P), and B2 microglobulin (B2M). In the 2 phases, HD*6 and HF*6, respectively, the weekly urea Kt was: 120 vs. 60 L; std Kt/V: 3.30 vs. 2.0; npcr: 1.26 vs. 1.42 g kg–1 day–1. Mass transfer/week Predialytic levels/L HD*6 HF*6 HD*6 HF*6 U (mmol) 2366 2204 23.2 38.5 C (µmol) 67240 51096 769.5 934.8 P (mmol) 127 111.5 1.91 2.34 B2M (mg) 311 763.5 34.2 30.16 In HF*6, predialysis solute concentration increased and weekly small molecule mass transfer was insufficient. B2M mass transfer was significantly increased and predialytic plasma concentration decreased, showing a higher clearance of middle molecules with convective transfer. The clinical tolerance was excellent during the two phases of treatment. This study allows to determine the optimal conditions for adequate daily HF: higher QB and exchange volume. Further studies with QB of at least 300 mL/min and with exchange volume of 15 L/session seem to be necessary.

Development of a Porous Silicon Product for Small Molecule Mass Spectrometry

ABSTRACTPrevious work has demonstrated the utility of desorption/ionization on silicon (DIOS) time-of-flight mass spectrometry (TOFMS) in drug molecule and peptide detection[1-7]. In this work, the utility of DIOS for small molecule detection is established using commercially available porous silicon (por Si)-based target plates for MS. Since the morphology and handling of the substrates can have dramatic effects on the MS characteristics, the development of consistent manufacturing methods and characterization protocols has been central to the production of reproducible target plates[7]. Using sample substrates manufactured in-house, we show that 1) small molecules and protein digests were detected without matrix-related peaks, 2) por Si morphology was optimized for small molecule detection, 3) reproducible DIOS plates were produced, 4) although the target plates were shown to be sensitive to contamination, a consistent cleaning procedure was developed to remove contaminants, and 5) stability and shelf life were characterized as a function of surface derivatization. Dynamic range, sensitivity, quantitation, speed of analysis, solution composition, and automated deposition have also been evaluated and are described in related work[7-9]. Potential applications include high-throughput small molecule assays for drug discovery[10a] and high sensitivity (sub-femtomole) peptide identification for proteomics[10b].