High-Sensitivity Elemental Mass Spectrometry of Fluorine by Ionization in Plasma Afterglow.

Fluorine elemental analysis using inductively coupled plasma mass spectrometry (ICPMS) is challenging because of low F ionization efficiency in the plasma and severe isobaric interferences. Notably, there is an increasing demand for ppb level fluorine measurements due to the rising importance of fluorinated compounds in pharmaceutical, environmental, and food analyses. Here, we report a new elemental ionization method where fluorinated analytes are introduced into an ICP to produce NaF followed by Na2F+ formation in the atmospheric-pressure plasma afterglow. The new method offers over 2 orders of magnitude improved sensitivities (180-500 cps/ppb versus 1.6-3.2 cps/ppb) for F detection. This approach also yields compound-independent F response for quantitation without compound-specific standards. Detection limits of ∼50 ppb F are attained using a single-quadrupole instrument without discrimination against isobaric interferences. Similar LODs are achievable only by isobaric interference reduction in ICPMS/MS. Importantly, the new approach offers facile interfacing to molecular MS instruments where LODs can be further improved via MS/MS and high-resolution MS techniques. The tolerance to matrix is demonstrated by quantitation of fluoride in infant formula, yielding recoveries of 86%-98% with repeatabilities of 3.5-6.3 RSD%.

A method for assessing and maintaining the reproducibility of mass spectrometric analyses of complex samples.

Direct injection mass spectrometric analysis of biological samples is potentially an attractive approach to the discovery of diagnostic patterns for specific pathophysiological conditions because of its speed and simplicity. Despite the possible benefits offered by such a method, its extensive application has been limited so far by several factors, including the inadequate reproducibility of the analytical results. We describe a method for monitoring and optimizing the performance of mass spectrometers used for biomarker discovery studies, based on the analysis of patterns of standardized spectral features. The method was successfully applied to maintaining spectral reproducibility during a multi-day analysis of hundreds of serum samples despite an ion source failure, which necessitated minor maintenance. The monitoring method allowed the early detection of that failure and the restoration of the spectral profiles after the system was restarted.

A method for monitoring and controlling reproducibility of intensity data in complex electrospray mass spectra: a thermometer ion-based strategy.

Reproducibility in mass spectral data is important in both biomarker discovery and spectral database searching. We report a strategy, employing a series of substituted benzylpyridinium thermometer ions that can be used to monitor changes in performance of multiple aspects of an electrospray ionization source that impact the intensity axis of a spectrum. Performance attributes, which could confound even isotope-based quantification strategies, are readily assessed using a mixture of thermometer ions. Based on the observed behavior of the ions, a procedure is proposed for monitoring instrument performance and compensating for factors that affect reproducibility across both time and instruments.

Chemical reaction interface mass spectrometry with high efficiency nebulization.

A high efficiency nebulizer (HEN) coupled to a heated spray chamber and a membrane desolvator is used for liquid sample introduction in chemical reaction interface mass spectrometry (CRIMS). Compared to the conventional thermospray nebulizer operated at solvent flow rate of 1 mL/min, the HEN provides small droplets at lower flow rates (10-100 microL/min), improving the desolvation and analyte transport efficiency. As a result, the sensitivity for carbon detection by CRIMS is improved by a factor of 4. The new arrangement offers an easy-to-use and robust interface, facilitating the availability of a variety of liquid chromatographic techniques to the CRIMS. Separation and detection of labeled peptides in a mixture of unlabeled biopolymers is illustrated at a solvent flow rate of 45 microL/min as an example of new possibilities offered by the improved liquid introduction interface.

The role of esterification on detection of protonated and deprotonated peptide ions in matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS).

Esterification was used to investigate how introduction of aliphatic chains within the peptide structure affects the MALDI response of ions analyzed in both polarity regimes. In binary mixtures containing equimolar amounts of a peptide with its correspondent alkyl ester, derivatization of the carboxylic groups has the tendency to increase MALDI detection of the modified protonated peptide ions. This positive effect on ion yield is more pronounced when longer alcohols are employed. In negative mode, the situation is antithetic and esterification produces a deleterious effect on the ion yield of the corresponding deprotonated species. From the data reported here we postulate that modifications of the acidic character of peptides prevent formation of anionic species under MALDI analysis. Furthermore, suppression of the formation pathway for anions alters the overall number of molecules which can undergo protonation. This results in an increased ion yield for the protonated esters.

Kinetic isotope effects in Ras-catalyzed GTP hydrolysis: evidence for a loose transition state.

A remote labeling method has been developed to determine (18)O kinetic isotope effects (KIEs) in Ras-catalyzed GTP hydrolysis. Substrate mixtures consist of (13)C-depleted GTP and [(18)O,(13)C]GTP that contains (18)O at phosphoryl positions of mechanistic interest and (13)C at all carbon positions of the guanosine moiety. Isotope ratios of the nonvolatile substrates and products are measured by using a chemical reaction interface/isotope ratio mass spectrometer. The isotope effects are 1.0012 (0.0026) in the gamma nonbridge oxygens, 1.0194 (0.0025) in the leaving group oxygens (the beta-gamma oxygen and the two beta nonbridge oxygens), and 1.0105 (0.0016) in the two beta nonbridge oxygens. The KIE in the beta-gamma bridge oxygen was computed to be 1.0116 or 1.0088 by two different methods. The significant KIE in the leaving group reveals that chemistry is largely rate-limiting whereas the KIEs in the gamma nonbridge oxygens and the leaving group indicate a loose transition state that approaches a metaphosphate. The KIE in the two beta nonbridge oxygens is roughly equal to that in the beta-gamma bridge oxygen. This indicates that, in the transition state, Ras shifts one-half of the negative charge that arises from P(gamma)-O(beta-gamma) fission from the beta-gamma bridge oxygen to the two beta nonbridge oxygens. The KIE effects, interpreted in light of structural and spectroscopic data, suggest that Ras promotes a loose transition state by stabilizing negative charge in the beta-gamma bridge and beta nonbridge oxygens of GTP.

A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms.

Blood-feeding pathogens digest hemoglobin (Hb) as a source of nutrition, but little is known about this process in multicellular parasites. The intestinal brush border membrane of the canine hookworm, Ancylostoma caninum, contains aspartic proteases (APR-1), cysteine proteases (CP-2), and metalloproteases (MEP-1), the first of which is known to digest Hb. We now show that Hb is degraded by a multi-enzyme, synergistic cascade of proteolysis. Recombinant APR-1 and CP-2, but not MEP-1, digested native Hb and denatured globin. MEP-1, however, did cleave globin fragments that had undergone prior digestion by APR-1 and CP-2. Proteolytic cleavage sites within the Hb alpha and beta chains were determined for the three enzymes, identifying a total of 131 cleavage sites. By scanning synthetic combinatorial peptide libraries with each enzyme, we compared the preferred residues cleaved in the libraries with the known cleavage sites within Hb. The semi-ordered pathway of Hb digestion described here is surprisingly similar to that used by Plasmodium to digest Hb and provides a potential mechanism by which these hemoglobinases are efficacious vaccines in animal models of hookworm infection.

In vitro performance of hemodialysis membranes after repeated processing.

BACKGROUND: Dialyzers reprocessed with chlorine-based solutions have been associated with increases in ultrafiltration coefficient and middle-molecule removal. Increased pore size has been hypothesized as the mechanism for the latter phenomenon. Dialyzers exposed to Amukin-D (Amuchina Int Inc, Gaithersburg, MD), a chlorine-based reprocessing agent, were evaluated for changes in molecular weight (MW) cutoff and ultrafiltration properties. METHODS: In vitro MW cutoff studies were performed on Fresenius F-80A (Fresenius, Lexington, MA) and Gambro Polyflux 17 (Gambro, Lakewood, CO) hemodialyzers that were reprocessed 20 times using Amukin-D. Permeability (Uf-A), defined as the area from the ultrafiltered compartment (Uf) compared with the area from the equivalent arterial compartment (A), for dextran across the hemodialyzer membrane was determined after the initial use and after reuses 1, 5, 10, 15, and 20 by using size-exclusion chromatography. RESULTS: Uf-A for dextran increased approximately 10-fold between hemodialyzer reuses 1 and 5. Thereafter, additional reprocessing did not increase the Uf-A ratio further. MW cutoff increased during these 5 washes and did not change thereafter. CONCLUSION: Reprocessing with Amukin-D increased the MW cutoff and permeability of both hemodialyzers between reuses 1 and 5, resulting in a greater ultrafiltration rate and greater middle-molecule removal. After reuse 5, there were no further increases in MW cutoff with additional reprocessing in either hemodialyzer. This suggests that reprocessing and storage of each hemodialyzer with Amukin-D affects the permeability of dextran in a nonlinear fashion and to a finite level, such that subsequent reprocessing has no further effect on the MW cutoff of the membrane.

Size-exclusion chromatography in multidimensional separation schemes for proteome analysis.

Size-exclusion chromatography (SEC) is a separation technique with a relatively low resolving power, compared to those usually utilized in proteomics. Therefore, it is often overlooked in experimental protocols, when the main goal is resolving complex biological mixtures. In this report, we introduce innovative multidimensional schemes for proteomics analysis, in which SEC plays a practical role. Liquid isoelectric focusing (IEF) was combined with SEC, and experimental results were compared to those obtained by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), well-established techniques relying upon similar criteria for separation. Additional experiments were performed to evaluate the practical contribution of SEC in multidimensional chromatographic separations. Specifically, we evaluated the combination of SEC and ion exchange chromatography in an analytical scheme for the mass spectrometric analysis of protein-extracts obtained from bacterial cultures grown in stable isotope enriched media. Experimental conditions and practical considerations are discussed.