Metal-ion binding and limited proteolysis of betabellin 15D, a designed beta-sandwich protein.

Betabellin 15D is a 64-residue, disulfide-bridged homodimer. When folded into a beta structure, the protein is predicted to have two clusters of three histidine residues, each cluster able to bind a divalent metal ion. When the protein was incubated with Cu2+, Zn2+, Co2+, or Mn2+, metal complexes of betabellin 15D were observed by electrospray-ionization mass spectrometry.

Analysis of macromolecules using nanoelectrospray ionization mass spectrometry and low-energy collision activation.

The mass spectrometric analysis of several proteins using nanoelectrospray (nanoES) with elastic collisions showed an improvement in the sensitivity over nanoES without collisional activation. We believe this effect is due to a better declusterization/ionization process. Optimization of the collision parameters can be easily performed during the long experiment time allowed using the nanoES source.

A multidimensional approach to protein characterization.

When mass spectrometry (MS) is used to study protein primary structure, it is used in a "static" mode. That is, the information is derived from a single MS or MS-MS spectrum. Information about more complex protein structure or protein interactions can also be gained via MS.

Comprehensive on-line LC/LC/MS of proteins.

This is a description of a comprehensive two-dimensional liquid chromatography (LC) system for the separation of protein mixtures. This system uses cation-exchange chromatography followed by reversed-phase chromatography (RPLC). The two LC systems are coupled by an eight-port valve equipped with two storage loops and under computer control.

Activation of a novel calcium-dependent protein-tyrosine kinase. Correlation with c-Jun N-terminal kinase but not mitogen-activated protein kinase activation.

Many G protein-coupled receptors (e.g. that of angiotensin II) activate phospholipase Cbeta, initially increasing intracellular calcium and activating protein kinase C.

DNA adduct formation in B6C3F1 mice and Fischer-344 rats exposed to 1,2,3-trichloropropane.

1,2,3-Trichloropropane (TCP) is a multispecies, multisite carcinogen which has been found to be an environmental contaminant. In this study, we have characterized and measured DNA adducts formed in vivo following exposure to TCP. [14C]TCP was administered to male B6C3F1 mice and Fischer-344 rats by gavage at doses used in the NTP carcinogenesis bioassay.

Specific and nonspecific dimer formation in the electrospray ionization mass spectrometry of oligonucleotides.

Specific and nonspecific noncovalent dimer ions of oligonucleotides (ODNs) were observed when mixtures of complementary or noncomplementary strands were analyzed via negative ion electrospray ionization mass spectrometry. Dimer formation was concentration dependent and nearly always occurred when the concentration of ODN exceeded 100 µM. Dimers were observed even for short-length ODNs for which the melting temperature (T m) was well below the experimental temperature and which, therefore, would not be expected to form stable solution duplexes.

Deuterium exchange of alpha-helices and beta-sheets as monitored by electrospray ionization mass spectrometry.

Deuterium exchange was monitored by electrospray ionization mass spectrometry (ESI-MS) to study the slowly exchanging (hydrogen bonded) peptide hydrogens of several alpha-helical peptides and beta-sheet proteins. Polypeptides were synthetically engineered to have mainly disordered, alpha-helical, or beta-sheet structure. For 3 isomeric 31-residue alpha-helical peptides, the number of slowly exchanging hydrogens as measured by ESI-MS in 50% CF3CD2OD (pD 9.

The mass spectrometry of helical unfolding in peptides.

Two model peptides, melittin and a growth hormone releasing factor (GRF) analog, have been studied by mass spectrometry and tandem mass spectrometry during the course of their deuterium exchange. Both peptides are known from previous work to form α-helices in solution. When the peptides are exposed to deuterated solvents, their masses increase as deuterium atoms replace protons in the exchangeable sites of the peptides.

Preliminary X-ray diffraction studies of recombinant 19 kDa human fibroblast collagenase.

Crystals of the catalytic domain of human fibroblast collagenase have been grown in the presence and absence of an inhibitor. Crystals of the inhibitor complex grew from 0.2 M ammonium sulfate and 15 to 30% PEG 8000 at 22 degrees C as bipyramids in the space group P6(2) or P6(4).

Conformation of cytochrome c studied by deuterium exchange-electrospray ionization mass spectrometry.

Deuterium exchange of bovine cytochrome c has been monitored by electrospray ionization mass spectrometry. Different charge-state distributions in the mass spectrum appear to represent different protein conformations, but rapid interconversion of the conformations can lead to a coincidence of the deuterium exchange rates. When interconversion is blocked, the conformation corresponding to higher m/z (lower charge) exchanges more slowly, indicating a tightly folded state.

Probing the helical content of growth hormone-releasing factor analogs using electrospray ionization mass spectrometry.

A series of growth hormone-releasing factor analogs have been studied by both circular dichroism and electrospray ionization mass spectrometry (ESI/MS). The peptides are 32 residues long and are known to adopt a random-coil structure in aqueous solution but become increasingly helical as the proportion of organic solvent is increased. Deuterium exchange was observed as an increase in mass of the peptide, as measured by ESI/MS.

Comparison of separation and detection techniques for human growth hormone releasing factor (hGRF) and the products derived from deamidation.

Separation of the deamidation products, Asp8 Leu27 hGRF(1-32)NH2 (MH+ = 3654) and isoAsp8 Leu27 hGRF(1-32)NH2 (MH+ = 3654), from the parent analogue Leu27 hGRF(1-32)NH2 (MH+ = 3653) was achieved by reversed-phase LC and CE, where the retention order was seen to change from tr isoAsp8 hGRF < tr Asn8 hGRF < tr Asp8 hGRF to tr Asn8 hGRF < tr Asp8 hGRF < tr isoAsp8 hGRF, respectively. Both reversed-phase LC and CE gave adequate separations, limits of detection and standard curves. However, CE was preferred due to shorter analysis time, better separation and a smaller demand for material.

Crystallization and preliminary X-ray diffraction studies of recombinant human interleukin-5.

Recombinant human interleukin-5 (rhIL-5) has been crystallized by the hanging drop vapor diffusion method using 0.1 M-Tris.HCl buffer (pH 8.

Human interleukin-5 expressed in Escherichia coli has N-terminal modifications.

Recombinant human interleukin-5 exists as four major isoforms all possessing N-terminal methionine. Peptide mapping and subsequent analysis by fast-atom-bombardment mass spectrometry (f.a.

Identification and quantitation of tetrapeptide deamidation products by mass spectrometry.

A method to quantify asparagine (Asn), aspartate (Asp) and isoaspartate (isoAsp) residues in small peptides by fast atom bombardment mass spectrometry (FAB-MS) was developed. Discrimination of isoAsp from Asp residues was accomplished by selective derivatization of isoAsp residues in acetic anhydride, D2O and pyridine. Deuteration occurred at any carbon adjacent to a free alpha-carboxyl group, through a transient oxazalone intermediate, allowing the isoAsp side chain and the C-terminus to incorporate deuterium.

Sequencing and characterization of the soybean leaf metalloproteinase : structural and functional similarity to the matrix metalloproteinase family.

A novel zinc endoproteinase has been sequenced and characterized from soybean leaves (Glycine max var Williams 82) and has been designated as Protein Identification Resource accession No. A41820 SMEP1 (soybean metalloendoproteinase 1). Comparison of the primary amino acid sequence with other zinc proteinases revealed the enzyme to be a new member of the matrix metalloproteinase (MMP) family of enzymes.

Mercury in a commercial preparation of rat amylin.

The biological activity of amylin is reported to vary widely depending on the source and purity of the material. Three commercial samples of rat amylin were compared for structural differences. The samples were nearly identical using most of the available analytical measures--amino acid analysis, HPLC retention, even Edman sequencing data.

Rapid optimization of enzyme substrates using defined substrate mixtures.

A strategy is described for the rapid optimization of kcat/Km for protease substrates. Selected positions of a given peptide substrate sequence are varied through synthesis with mixtures of amino acids. Incubation of the resulting peptide mixture with the enzyme of interest and analysis by high pressure liquid chromatography provides a direct measure of analogs with enhanced kcat/Km.

Structure of Saccharomyces cerevisiae mating hormone a-factor. Identification of S-farnesyl cysteine as a structural component.

Mating type a cells of the yeast Saccharomyces cerevisiae produce a mating hormone, the a-factor, that we have previously characterized as a very hydrophobic, modified dodecapeptide (Betz, R., Crabb, J. W.

Correction of the cDNA-derived protein sequence of prostatic spermine binding protein: pivotal role of tandem mass spectrometry in sequence analysis.

Spermine binding protein (SBP) is a rat ventral prostate protein that binds various polyamines, and the level of this protein and its mRNA is regulated by androgens. Previously, the cDNA for SBP was cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from cloned and sequenced and an amino acid sequence deduced from the cDNA.

Isolation and identification of the principal siderophore of the dermatophyte Microsporum gypseum.

The dermatophyte Microsporum gypseum has been shown to produce two siderophores under conditions of low-iron stress. These compounds have been separated as Fe(III) complexes on silica gel, and the principal siderophore has been identified as ferricrocin using the methods of amino acid analysis, comparative thin-layer chromatography, partial sequencing by gas chromatography-mass spectrometry, ultraviolet spectroscopy, and proton nuclear magnetic resonance spectroscopy of the Al(III) complex.

Negative ion chemical ionization mass spectrometry of 2,4-dinitrophenyl amino acid esters.

We report the negative ion chemical ionization mass spectra of 2,4-dinitrophenyl (DNP) amino acid methyl esters. For the common amino acids, these derivatives exhibit very simple mass spectra; the molecular anion is the base peak in all cases. The electrophilicity of the DNP group allows selective and sensitive ionization.

Advances in gas chromatographic mass spectrometric protein sequencing. 2--Application to membrane proteins.

The primary structure of the integral membrane protein bacteriorhodopsin was determined by an efficient combination of gas chromatographic mass spectrometric techniques with the Edman degradation. This combination of methodologies circumvented many of the experimental difficulties associated with the insolubility of bacteriorhodopsin and its primary degradation fragments in aqueous buffers. Specifically, in the gas chromatographic mass spectrometric analysis of the cyanogen bromide peptides derived from bacteriorhodopsin, it has been possible to identify homoserine-containing peptides which served as a starting point for the construction of C-terminal sequences.