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R C Beavis

Publications and source records attributed to R C Beavis.

29 records · Page 2Linked to original sources
PubMed1993

The extracellular hemoglobin of the earthworm, Lumbricus terrestris. Determination of subunit stoichiometry.

The giant extracellular hemoglobin of the earthworm, Lumbricus terrestris, has four major O2-binding chains, a, b, and c (forming a disulfide-linked trimer) and d ("monomer"). Participation of additional "linker" chains L1, L2, and L3 is necessary for the assembly of the approximately 3,900+ kDa two-tiered hexagonal structure. We have determined the proportions of linker chains, trimer, and chain d in the hemoglobin by reverse phase high performance liquid chromatography which resolves all of the components and also permits simultaneous determination of the heme content. The proportions of components were determined by two independent procedures: integration of the absorbance peaks at 220 nm and amino acid analysis of the peak fractions. The results indicate that the weight proportion of linker chains is 0.163 +/- 0.023. This value, together with molecular masses determined both by amino acid sequence analysis and by matrix-assisted laser desorption mass spectrometry, gives a molar ratio of abcd chains to linkers of 8:1, corresponding to the minimal unit (abcd)2.L. This ratio suggests that 24 (abcd)2 units and 24 linker chains form the complete structure with a total calculated mass of polypeptide of 3,975 kDa with hemes on chains a, b, c and d and on one linker. The calculated heme content is 3.1% not including carbohydrate. This accounts for a measured heme content of 3.0% on a polypeptide basis. Additional mass (approximately 133 kDa, 3.4%), attributed to carbohydrate, brings the total mass to 4,108 kDa with a minimum molecular mass/heme of 20,500 Da. The presence of equimolar quantities of three unique linker chains means that the apparent one-twelfth structural units seen by electron microscopy cannot all be identical.

Amino Acid Sequence↗
PubMed1992

Heterodimeric structure of the spider toxin omega-agatoxin IA revealed by precursor analysis and mass spectrometry.

We report the first molecular characterization of a precursor sequence for a small, Ca2+ channel blocking, peptide spider toxin, omega-agatoxin IA. By integrating information generated from a molecular genetic approach using agatoxin cDNAs with data provided from mass spectrometry of the mature toxin, we were able to deduce the likely mechanisms by which the toxin precursor peptide is processed to its mature heterodimeric form. A particularly interesting feature of the prepropeptide is the occurrence of two glutamate-rich sequences interposed between the signal sequences, the major peptide toxin, and the minor toxin peptide. Excision of the more distal glutamate-rich region appears to be signaled by flanking arginine residues but likely occurs only after a disulfide linkage has formed between the major and minor chains of the mature toxin. Our molecular genetic approach toward characterizing this toxin will allow us to quickly generate a series of spider sequences from which mature toxin structures can be deduced and eventually expressed. Additionally, this approach will provide insights into the evolutionary divergence observed among spider peptide toxins.

Agatoxins↗
PubMed1991

Preparation, properties, and plasma retention of human hemoglobin derivatives: comparison of uncrosslinked carboxymethylated hemoglobin with crosslinked tetrameric hemoglobin.

Human hemoglobin A has been crosslinked by diisothiocyanatobenzenesulfonate to give a limited number of products in a yield of approximately 70%. The predominant product was crosslinked between subunits within a tetramer and had a Mr of 64,000; no higher Mr species were formed. This product had one crosslink per tetramer located between the NH2 termini of its alpha chains, as established by HPLC analysis, amino acid analysis, Edman degradation, and mass spectrometry. This crosslinked derivative had a slightly increased oxygen affinity [P50 = 9 mmHg (1 mmHg = 133 Pa); P50 for unmodified hemoglobin = 11 mmHg], and the retention time of this derivative in the circulation of rats was 2.9 and 3.3 hr at two hemoglobin concentrations (7 g/dl and 14 g/dl, respectively). The half-life of an uncrosslinked carboxymethylated derivative, which has a low oxygen affinity (P50 = 28 mmHg), was 0.6 and 0.7 hr under the same conditions. Therefore, prolongation of the plasma-retention time of infused hemoglobin is dependent on the crosslinking of the tetramer but independent of the oxygen affinity of the derivative.

Allosteric Regulation↗
PubMed1990

High-accuracy molecular mass determination of proteins using matrix-assisted laser desorption mass spectrometry.

A method for obtaining protein molecular masses with an accuracy of approximately +/- 0.01% by matrix-assisted laser desorption using an internal calibrant is described. The technique allows accurate mass determinations of protein sample sizes as small as 1 pmol. High concentrations of organic and inorganic contaminants (e.g. 1 M urea) do not strongly affect either the signal intensity or the mass assignment. The ability to assign an accurate molecular mass to a protein is contingent on the observation of clearly resolved protonated molecule ions in the mass spectrum.

Animals↗
PubMed1990

Rapid, sensitive analysis of protein mixtures by mass spectrometry.

We have developed a method for determining the molecular masses of proteins in complex mixtures by mass spectrometry. The method has the capacity to examine the components of mixtures without using any chromatographic separation steps and will tolerate relatively large amounts of buffers and inorganic contaminants. It allows the simultaneous determination of protein molecular masses from 1 to 40 kDa with an accuracy of +/- 0.01% and above 40 kDa with reduced accuracy. The lower limit for practical detection of a protein is a concentration of approximately 0.1 microM, and less than 1 microliter of such a solution is consumed. The analysis is very fast: less than 15 min is necessary to perform the complete analysis, including sample preparation, introduction into the mass spectrometer, mass spectrum collection, and data reduction. The mass spectrum that is obtained does not require elaborate interpretation because there is no fragmentation of the ionized protein (or protein subunit) molecule. Therefore, there is a one-to-one correspondence between the peaks in the mass spectrum and the proteins present in the original mixture. The spectra assume the appearance of chromatograms, with the abscissa being mass-to-charge ratio rather than chromatographic retention time.

Animals↗
PubMed1989

Factors affecting the ultraviolet laser desorption of proteins.

The production of high-mass quasimolecular ions from proteins by matrix-assisted ultraviolet laser desorption is described. A simple time-of-flight system using a Q-switched frequency-quadrupled Nd-YAG laser to desorb protein molecules is shown to have a mass range of up to 116,000 u by the observation of intact, singly charged quasimolecular ions from 700 fmol of beta-galactosidase subunit (mol.wt = 116,336 Da). Both positive- and negative-ion spectra of proteins are shown. Four new matrix materials, with properties as good as or better than nicotinic acid, are described. A mass resolution of approximately 500 (full width at half maximum definition) is demonstrated for proteins with mol.wt less than 20,000 Da. Product species, formed by fast photochemical reactions in the matrix, are observed to form adduct ions with protein molecules. These adduct ions are a significant cause of the observed broadness of protein quasimolecular ion peaks. The practical physical considerations in detection of large-mass quasimolecular ions from laser desorption, such as detector overloading, are discussed.

Animals↗
PubMed1989

Cinnamic acid derivatives as matrices for ultraviolet laser desorption mass spectrometry of proteins.

The paper reports the discovery of three new matrices for the matrix-assisted laser desorption of proteins. These new matrices (sinapinic, ferulic and caffeic acids) are cinnamic acid derivatives that have several practical advantages over the nicotinic acid matrices previously used. These materials form much less intense photochemically generated adduct peaks in the protein quasimolecular ion signal and the adduct peaks that are present are easier to resolve. These matrices produce intense protonated-molecule ions from all of the proteins (over 50) so far examined. These new matrices are also very stable in a vacuum, allowing for their convenient use in very high vacuum applications (e.g., Fourier transform ion cyclotron resonance mass spectrometry).

Animals↗
PubMed1989

Matrix-assisted laser-desorption mass spectrometry using 355 nm radiation.

Matrix-assisted laser desorption of proteins with the 355 nm frequency-tripled output of a neodymium:yttrium-aluminium-garnet laser has been demonstrated. In order to produce desorption at this wavelength, the cinnamic acid derivatives ferulic, caffeic and sinapinic acids have been employed as matrices. Excellent sensitivity (routinely one pmol of protein) and good mass resolution (m/delta m 400, Full width at half maximum) have been obtained by this new method. Ultraviolet photo-spectrometry of the cinnamic acid derivative matrices suggests strongly that they can be used as protein laser-desorption matrices at any wavelength between 260 nm and 360 nm, allowing the development of laser-desorption mass spectrometers based on nitrogen or excimer lasers.

Caffeic Acids↗
PubMed1986

Automated dry fraction collection for microbore high-performance liquid chromatography-mass spectrometry.

A method is presented for the collection of dry fractions from a microbore high-performance liquid chromatographic column. These fractions are electrosprayed onto a foil strip that is being moved past the spray in steps. These solid deposits are in a form which is compatible with solid-phase secondary-ion mass spectrometry, in particular the time-of-flight instrument that has been developed in our laboratory. Because the type of ionization used in static secondary-ion spectrometry is essentially non-destructive, the non-volatile eluent is available for any other analytical method after mass analysis. The chromatogram of a mixture of peptides was re-constructed from the mass spectra of fractions collected in this way. This chromatogram is shown and its features are examined.

Buffers↗
PubMedDate not supplied

Implementation of an algorithm for modeling disulfide bond patterns using mass spectrometry.

The paper describes the implementation of a software system based on the Fenyö disulfide bond assignment algorithm. The system allows an investigator to enter data derived from mass spectrum peak assignments, a target protein sequence and other experimental conditions. The output of the system is the set of disulfide bonding pattern models that are consistent with the experimental evidence. The software and code are available through a public web site, which also has a functioning, publicly accessible version of the disulfide bond modeler. This implementation was tested as part of a project to check homology-based assignments disulfide bonding patterns of human integrins.

Algorithms↗