Peptide mass mapping constrained with stable isotope-tagged peptides for identification of protein mixtures.

Through proteolysis and peptide mass determination using mass spectrometry, a peptide mass map (PMM) can be generated for protein identification. However, insufficient peptide mass accuracy and protein sequence coverage limit the potential of the PMM approach for high-throughput, large-scale analysis of proteins. In our novel approach, nonlabile protons in particular amino acid residues were replaced with deuteriums to mass-tag proteins of the S. cerevisiae proteome in a sequence-specific manner. The resulting mass-tagged proteolytic peptides with characteristic mass-split patterns can be identified in the data search using constraints of both amino acid composition and mass-to-charge ratio. More importantly, the mass-tagged peptides can further act as internal calibrants with high confidence in a PMM to identify the parent proteins at modest mass accuracy and low sequence coverage. As a result, the specificity and accuracy of a PMM was greatly enhanced without the need for peptide sequencing or instrumental improvements to obtain increased mass accuracy. The power of PMM has been extended to the unambiguous identification of multiple proteins in a 1D SDS gel band including the identification of a membrane protein.

Speciation of gold(III)-L-histidine complex: a multi-instrumental approach.

The formation and structure of gold (III)-L-histidine complex was investigated with the use of carbon (13C) and proton (1H) nuclear magnetic resonance (NMR), infrared (IR) spectroscopy, capillary electrophoresis (CE), capillary electrophoresis-inductively coupled plasma-mass spectroscopy (CE-ICP-MS), X-ray fluorescence spectroscopy (XRF), matrix-assisted laser desorption/ionization (MALDI), and laser desorption mass spectroscopy (LDMS). It was found that two L-histidine molecules and one gold ion slowly form a complex in acidic solution. Each L-histidine molecule provided two nitrogen ligands; one was the alpha-amino group and the other from the imidazole ring. The Au(III)-bis-L-histidine complex precipitates after deprotonation of the free carboxylic group, resulting in an increase in the solution acidity. Determination of the exact sequence of events and the identity of the complex was a comprehensive instrumental analysis problem involving the above techniques.

Stable-isotope-assisted MALDI-TOF mass spectrometry for accurate determination of nucleotide compositions of PCR products.

In parallel with a large-scale sequencing effort, the human genome project will need next-generation tools for accurate and efficient analyses of the enormous pool of DNA sequences. Such analyses are required for (a) validation of DNA sequences, (b) comparison of a parent (known) sequence with a related (unknown) sequence, and (c) characterization of sequence polymorphisms in various genes, especially those associated with genetically inherited human diseases. Here, we report a novel method that combines stable isotope 13C/15N labeling of PCR products of the target sequences with analysis of the mass shifts by mass spectrometry (MS). The mass shift due to the labeling of a single type of nucleotide (i.e., A, T, G, or C) reveals the number of that type of nucleotide in a given DNA fragment. Using this technique, we have accurately determined nucleotide compositions of DNA fragments. The method has also been applied to score a known single-nucleotide polymorphism (SNP). The comparisons of nucleotide compositions determined by our method among homologous sequences are useful in sequence validation, sequence comparison, and characterizations of sequence polymorphisms.

Glow discharge source interfacing to mass analyzers: theoretical and practical considerations.

The fundamental requirements for the optimum mechanical interface between a glow discharge ion source and a mass spectrometer are described in this paper. Specifically, the properties of a typical glow discharge ion source are compared and contrasted to those of a typical inductively coupled plasma ion source. The critical parameters and theoretical considerations of glow discharge and inductively coupled plasma ion source interfaces are reviewed, and the results of experiments using both quadrupole and time-of-flight mass spectrometers with a glow discharge source are presented. The experimental results clarify several important problems in the glow discharge ion sampling process. Our findings indicate that a shock wave structure does not occur in the supersonic expansion of the glow discharge ion source. Ions of different masses have similar initial kinetic energies in the glow discharge; thus, the angle of the skimmer cone is not a critical parameter for efficient ion beam extraction. Another consquence is that space charge effects in glow discharge ion sources repel heavy ions farther off axis than light ions. Thus, there are distinct and fundamental differences between glow discharge and inductively coupled plasma ion sources which are relevant to both ion sampling and ion extraction processes.

X-ray ionization and fragmentation for use with time-of-flight mass spectrometry.

In this paper, we introduce laser desorption X-ray ionization for producing ions from the previously undetected neutral species present during laser desorption mass spectrometry. Studies involving the laser desorption of simple sugars were conducted to illustrate the differences between spectra with and without the X-ray source. Ionization was made possible by placing a 200 mCi Am X-ray source directly into the ionization chamber of a time-of-flight mass spectrometer.

Identification of metabolic intermediates in microbial degradation of phenol using laser desorption time-of-flight mass spectrometry.

Laser desorption time-of-flight (LD-TOF) mass spectrometry was utilized to determine the metabolic intermediates in microbial degradation of phenol. The identified components were in good agreement with the well-documented pathway. This technique also demonstrated excellent precision. Analytical merits, instrumentation and methodology are discussed.

Brain aluminum in Alzheimer's disease using an improved GFAAS method.

Aluminum (Al) has been determined in autopsy brain samples (20 mg, dry weight) from 10 histologically verified Alzheimer's disease (AD) patients and 10 neurologically normal controls using an improved graphite furnace atomic absorption method. The method makes use of a potassium dichromate matrix modifier to circumvent interferences to the Al determination by the high levels of phosphorus and alkali metals in brain tissue. Brain regions studied included middle frontal gyrus, hippocampus, inferior parietal lobule, and the superior and middle temporal gyri. Small, but significant (p < 0.05) elevations of Al were observed in AD hippocampus, inferior parietal lobule and superior and middle temporal gyri, compared to corresponding control tissues. In contrast to other reports in the literature, no extremely high Al levels (> 20 micrograms/g, dry weight) were observed in our samples. Our highest value for Al in AD brain was 8.0 micrograms/g. These results suggest that there is a small focal increase of Al in specific regions of AD brain compared to age-matched controls. The fact that the increase is small, coupled with the analytical difficulties of accurate bulk sample Al determinations by any technique in the brain matrix, may account for the discordant literature reports for Al in AD brain.

Evaluation of the metal uptake of several algae strains in a multicomponent matrix utilizing inductively coupled plasma emission spectrometry.

Three freshwater heat-killed, lyophilized blue-green algae strains have been characterized as to their ability to accumulate heavy metals with a focus on the utilization of these algae as an analytical preconcentration technique. This study examines the metal uptake in several multicomponent mixtures by using inductively coupled plasma optical emission spectrometry (ICP-OES). Six milligrams of a pure strain of algae was added to 20-mL aliquots of buffered (pH 5.5-6.5) multielement solutions containing 0.1, 0.5, 1.0, 2.0, and 4.0 mg/L of K, Mg, Ca, Fe, Sr, Co, Cu, Mn, Ni, V, Zn, As, Cd, Mo, Pb, and Se. All three algae strains exhibit relatively high adsorption affinities for Fe, Pb, and Cu, with uptake between 70 and 98% at the 4 ppm concentration level. Biosorption occurs for essentially every element with the relative affinities decreasing in the order Pb greater than Fe greater than Cu greater than Cd greater than Zn greater than Mn greater than Mo greater than Sr greater than Ni greater than V greater than Se greater than As greater than Co for Chlorella pyrenoidosa at the 4 mg/L concentration level. Although some minor differences were seen, the other algae strains (Stichococcus bacillaris and Chlamydomonas reinharti) displayed similar adsorption behavior over the concentration range studied, indicating similar cell wall binding sites. Langmuirian isotherms exhibited a minimum of two slopes over the concentration range of 0.1-4.0 mg/L, indicating the probable existence of at least two adsorption mechanisms.

Penicillin-resistant strains of Neisseria gonorrhoeae in Shahre-Now.

Of 921 women examined, 112, or 12.1%, were identified as having gonorrhoea by positive culture; 25.6% of 78 strains isolated from the women were resistant to penicillin at 0.10 mug/ml level and 14.1% resistant at the 0.50 mug/ml level as determined by an agar dilution MIC test. Our results were compatible with a similar but larger study reported in the USA by Jaffe et al. 1976.