Of methods for dissociation of multiply charged peptide and protein ions, electron capture dissociation (ECD) has the advantages of cleaving between a high proportion of amino acids, without loss of such posttranslational modifications as glycosylation and carboxylation. Here this capability is successfully extended to phosphorylation, for which collisionally activated dissociation (CAD) can cause extensive loss of H3PO4 and HPO3. As shown here, these losses are minimal in ECD spectra, an advantage for measuring the degree of phosphorylation. For phosphorylated peptides, ECD and CAD spectra give complementary backbone cleavages for identifying modification sites. For a 24-kDa heterogeneous phosphoprotein, bovine beta-casein, activated ion ECD cleaved 87 of 208 backbone bonds that identified a phosphorylation site at Ser-15, and localized three more among Ser-17,-18, -19, and -22 and Thr-24, and the last among four other sites. This is the first direct site-specific characterization of this key post-translational modification on a protein without its prior degradation, such as proteolysis.
Mass spectrometry (MS) has become an indispensable tool for peptide and protein structure analysis because of three unique capabilities that enable it to be used to solve structural problems not easily handled by conventional techniques. First, MS is able to provide accurate molecular weight information on low-picomole amounts of peptides and proteins independent of covalent modifications that may be present. Second, this information is obtainable for peptides present in complex mixtures such as those that result from a proteolytic digest of a protein. Third, by using tandem MS, partial to complete sequence information may be obtained for peptides containing up to 25 amino acid residues, even if the peptides are present in mixtures. Sensitivity and speed of the MS-based approaches now equal (and in some cases exceed) that of Edman-based sequence analysis. In this perspective we discuss how MS, tandem high-performance MS, and on-line liquid chromatography/MS using fast atom bombardment or electrospray ionization have been integrated with more conventional techniques in order to increase the accuracy and speed of peptide and protein structure characterization. The expanding role of matrix-assisted laser desorption MS in protein analysis is also described. The unique niche that MS occupies for locating and structurally characterizing posttranslational modifications of proteins is emphasized. Examples chosen from the authors' laboratory illustrate how MS is used to sequence blocked proteins, define N- and C-terminal sequence heterogeneity, locate and correct errors in DNA- and cDNA-deduced protein sequences, identify sites of deamidation, isoaspartyl formation, phosphorylation, oxidation, disulfide bond formation, and glycosylation, and define the structural class of carbohydrate at specific attachment sites in glycoproteins.
Three new imidazole alkaloids, leucettamines A [1] and B [2] and leucettamidine [31, have been isolated from the Palauan sponge Leucetta microraphis. Their structures were established on the basis of extensive spectral analyses. Leucettamine A showed potent leukotriene B4 receptor binding activity (K¡ =1.3 µ ), while leucettamine B was essentially inactive (K¡ = 100 µ ) and leucettamidine showed significant activity (K¡ = 5.3 µ ). With leucettamine A identified as a pure LTB4 receptor antagonist, a new structure lead is presented to inflammation therapy.
The pathway for the anaerobic catabolism of gallic acid by Eubacterium oxidoreducans was studied by using both in vivo and cell-free systems 16,300 x g and were washed at least once with either 100 mM potassium phosphate, pH 7.0, or 100 mM Tris hydrochloride, pH 7.5. Cell extracts were prepared by the passage of the cells, which were suspended in a small amount of buffer, once through a French pressure cell. When anaerobic conditions were required, buffers were sparged with nitrogen, and all manipulations were all done in an anaerobic glove box. 2-Mercaptoethanol (10 mM) was included in anaerobic buffers.Identification of pathway intermediates. Isolated intermediates were identified by gas chromatography-mass spectrometry (GC-MS) and, when indicated, by proton magnetic resonance (PMR) spectroscopy. For GC analysis, trimethylsilyl derivatives of phenolic compounds were prepared. Dried samples were dissolved in 0.1 ml of pyridine, and 0.1 to 0.2 ml of bis-trimethylsilyltrifluoroacetamide (plus 1% trimethyltrichlorosilane) was added. The samples were heated for 20 min at 60°C. Compounds were chromatographed on an OV-17 column with the temperature increasing from 110 to 270°C at a rate of 10°C/min. GC-MS was performed with a GC-MS spectrometer (5985B; HewlettPackard Co., Palo Alto, Calif.) with the CI-El source set at 70 eV and at 200°C. For PMR experiments, a Varian XL-200 nuclear magnetic resonance spectrometer was used. Underivatized samples were dissolved in D20, and derivatized samples were dissolved in CDCl3, with trimethylsilylpropionate and tetramethylsilane, respectively, as references.Enzyme determinations. All assays were done with a Response spectrophotometer (Gilford Instrument Laboratories, Oberlin, Ohio) at 37°C with 1-ml cuvettes containing 0.5 ml of reaction mixture. When assays were performed anaerobically, the cuvettes were filled in an anaerobic glove box under 95% N2-5% H2 and were sealed with a piece of gum-rubber tubing. After the cuvettes were prewarmed in the cuvette holder, a final addition of substrate or enzyme
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.