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.     

Further aspects of the roaming mechanism in formaldehyde dissociation

Recently we reported a novel “roaming” dissociation pathway of formaldehyde in which one of the H atoms strays far from the minimum energy reaction path, explores a broad region of the potential energy surface, then abstracts the remaining H atom to form molecular products, without going near the configuration of the conventional transition state saddle point. The detailed dynamics of the abstraction mechanism and its energy dependence have already been reported. Here, with a combination of experimental and theoretical results, we examine the roaming behavior at the energetic extremes. We show evidence of roaming below the threshold of the radical dissociation channel and consider the implications of this and the possible existence of a transition state for the roaming mechanism. We also show the occurrence of roaming up to 3200 cm⁻¹ above the threshold of the triplet dissociation channel. In addition, we present results affording deeper insight into the dynamics of the roaming mechanism: we show evidence of roaming leading to CO in v = 1, and examine the issue of nuclear spin conservation during dissociation via the roaming mechanism.     

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.