Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.     

Negative ion electrospray mass spectrometry of nucleotides: ionization from water solution with SF6 discharge suppression

The total current and selected ion currents from the electrospray ionization (ES1) of 10^?5 M solutions of cocaine hydrochloride and deoxycytidine monophosphate (dCMP) monosodium salt in methanol and water solvents were compared in positive and negative ion modes, respectively, without and with SF₆, gas as a discharge suppressant. The ESI onset voltages (V_on), were the same for the positive and negative ion modes. The V_on, for methanol was much lower than that for water and in agreement with the equation of D. P. H. Smith, who attributes the difference to the higher surface tension of water. The onset of electric discharge (V_dis) without SF₆, occurred at lower capillary voltages for the negative relative to the positive ion mode for methanol; but V_dis is much higher than V_on for methanol, and discharges do not interfere with ESI operation. For water, V_on ≈ V_dis in the absence of SF₆, in the negative ion mode, and ESI operation is impossible without SF₆, discharge suppression. The discharge problem in the positive ion mode is less severe, but SF₆, is still very useful. A dynamic range of 10 ^?7–10^?5 M was obtained by selected ion monitoring of [dCMP - H]^? at 4.5 and 20 μL/min. flows. Subpicomole detection limits for the nucleotide salt were obtained under these conditions.     

Electrospray mass spectrometry of methanol and water solutions suppression of electric discharge with SF6 gas

An equation by D. P. H. Smith predicts the capillary voltage required for the onset of electrospray (ES). For different solvents the voltage increases with the square root of the surface tension. Water requires a potential that is 1.8 times higher than that for methanol. This is verified experimentally. The higher potential required for water leads to ES in the presence of corona electric discharge. For low total ES plus corona currents, the electrosprayed analyte ion intensity is not adversely affected by the presence of discharge. At high total currents, there is a large decrease of analyte sensitivity. The sensitivity decrease is probably due to adverse space charge effect at high currents. The discharge can be suppressed by adding sulfur hexafluoride to the ambient gas. Both sensitivity and signal stability are improved. However, the sensitivity still remains lower by a factor of ≈ 4 relative to that observed with methanol. This is attributed to lower efficiency of gas-phase ion formation from charged water, relative to methanol, droplets.     

Hydrazine-mediated cyclization of Ugi products to synthesize novel 3-hydroxypyrazoles

This report discloses a novel concise synthesis of a series of 3-hydroxypyrazoles 5 via a tandem Ugi/debenzylation /hydrazine-mediated cyclization sequence. Herein, n-butyl isocyanide 4b was utilized as an alternative to classical convertible isocyanides enabling high yielding hydrazine-mediated cyclization. Taken together, a novel class of 3-hydroxypyrazoles 5a-5i was synthesized with potential to be of interest in future library enrichment strategies.     

Comparison of microbial receptor assay and liquid chromatography for determination of penicillin G and amoxicillin in milk powder

A microbial receptor assay (Charm II Tablet Beta-Lactam Test) and liquid chromatography (LC) were compared for determination of penicillin G (PG) and amoxicillin (AMOX) in reconstituted milk powder. Nonfat dry milk and whole dry milk were reconstituted (10%, w/v) to concentrations of 0, 2.5, 5, 7.5, and 10 ppb PG; nonfat dry milk was reconstituted (10%, w/v) to 0, 7.5, 10, and 15 ppb AMOX. Reconstituted samples were analyzed blindly by each method. Concentrations determined by both methods demonstrated good agreement. A significant difference between methods (p < or = 0.05) was observed only for 7.5 ppb PG in defatted dry milk. Significant differences were not observed between known concentrations and concentrations determined by the Charm II assay for PG or AMOX in defatted dry milk and PG in whole dry milk. Results by LC showed significant differences (p < 0.05) between known and measured concentrations at 10 ppb PG in both milks and 0 ppb AMOX in defatted dry milk. These results suggest that both the microbial receptor assay and LC may be useful for determination of PG and AMOX near safe level and tolerance, respectively, in reconstituted milk powder.     

Toward a stable apoptolidin derivative: identification of isoapoptolidin and selective deglycosylation of apoptolidin

[formula: see text] Isoapoptolidin was isolated from crude fermentation extracts of the apoptolidin-producing microorganism Nocardiopsis sp. Apoptolidin isomerizes to isoapoptolidin upon treatment with methanolic triethylamine to establish a 1.4:1 equilibrium mixture of isoapoptolidin and apoptolidin. Semisynthesis of a peracetylated and deglycosylated derivative of apoptolidin is also described.     

Molecular beam epitaxy

Molecular beam epitaxy (MBE) is a process for growing thin, epitaxial films of a wide variety of materials, ranging from oxides to semiconductors to metals. It was first applied to the growth of compound semiconductors. That is still the most common usage, in large part because of the high technological value of such materials to the electronics industry. In this process beams of atoms or molecules in an ultra-high vacuum environment are incident upon a heated crystal that has previously been processed to produce a nearly atomically clean surface. The arriving constituent atoms form a crystalline layer in registry with the substrate, i.e., an epitaxial film. These films are remarkable because the composition can be rapidly changed, producing crystalline interfaces that are almost atomically abrupt. Thus, it has been possible to produce a large range of unique structures, including quantum well devices, superlattices, lasers, etc., all of which benefit from the precise control of composition during growth. Because of the cleanliness of the growth environment and because of the precise control over composition, MBE structures closely approximate the idealized models used in solid state theory.

This discussion is intended as an introduction to the concept and the experimental procedures used in MBE growth. The refinement of experimental procedures has been the key to the successful fabrication of electronically significant devices, which in turn has generated the widespread interest in the MBE as a research tool. MBE experiments have provided a wealth of new information bearing on the general mechanisms involved in epitaxial growth, since many of the phenomena initially observed during MBE have since been repeated using other crystal growth processes. We also summarize the general types of layered structures that have contributed to the rapid expansion of interest in MBE and its various offshoots. Finally we consider some of the problems that remain in the growth of heteroepitaxial structures, specifically, the problem of mismatch in lattice constant between layers and between layer and substrate. The discussion is phenomenological, not theoretical; MBE has been primarily an experimental approach based on simple concepts.     

Extraction of osmium thiocyanate and its separation from ruthenium by polyurethane foam

A detailed investigation of the conditions for formation and extraction of the thiocyanate complex of osmium by polyether-type polyurethane foam is reported. The complex which formed in solution was extracted through the “cation-chelation” mechanism and distribution coefficients of more than 10⁴ were obtained. By using conditions which inhibit the formation of the osmium-thiocyanate complex, it was possible to leave 95% of osmium in the aqueous phase while extracting more than 95% of ruthenium into polyurethane foam.     

Distributed automated docking of flexible ligands to proteins: Parallel applications of AutoDock 2.4

Summary AutoDock 2.4 predicts the bound conformations of a small, flexible ligand to a nonflexible macromolecular target of known structure. The technique combines simulated annealing for conformation searching with a rapid grid-based method of energy evaluation based on the AMBER force field. AutoDock has been optimized in performance without sacrificing accuracy; it incorporates many enhancements and additions, including an intuitive interface. We have developed a set of tools for launching and analyzing many independent docking jobs in parallel on a heterogeneous network of UNIX-based workstations. This paper describes the current release, and the results of a suite of diverse test systems. We also present the results of a systematic investigation into the effects of varying simulated-annealing parameters on molecular docking. We show that even for ligands with a large number of degrees of freedom, root-mean-square deviations of less than 1 Å from the crystallographic conformation are obtained for the lowest-energy dockings, although fewer dockings find the crystallographic conformation when there are more degrees of freedom.The AutoDock 2.4 suite is written in ANSI C, and is supplied with Makefiles for the following platforms: Convex, DEC Alpha OSF/1, Hewlett-Packard Precision Architecture, Silicon Graphics, and Sun. The AutoDock suite of programs is freely available to the noncommercial scientific community and to educational establishments. Further information, including additional figures and MPEG animations showing all docked conformations for each test system, can be found at the following URL: http://www.scripps.edu/pub/olson-web/doc/autodock.     

Study of the lithium thiocyanate quadrupole by ultra-violet absorption spectroscopy

A spectroscopic method for studying quadrupole formation is described, and experimental results are presented for lithium thiocyanate in a tetrahydrofuran/hexane solvent system. From molar absorptivity equations and an equation by Fuoss and Kraus, a direct relationship is derived between the quadrupole formation constant, K_Q, and the dielectric constant of the medium. Experimental results are consistent with the equation and allow an estimate of the distance between lithium and thiocyanate in the ion pair.