Location of alkali metal binding sites in endothelin A selective receptor antagonists, cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) and cyclo(D-Trp-D-Asp-Pro-D-Ile-Leu), from multistep collisionally activated decompositions.

We previously showed by using mass spectrometry that endothelin A selective receptor antagonists BQ123 and JKC301 form novel coordination compounds with sodium ions. This property may underlie the ability of an ET(A) antagonist to induce net tubular sodium reabsorption in the proximal tubule cells and reverse acute renal failure induced by severe ischemia. We have now defined the metal binding sites on BQ123 and JKC301 by subjecting the metal-containing peptides to multiple stages of collisionally activated decomposition (CAD) in an ion trap mass spectrometer. When submitted to low-energy CAD, the ring opens at the Asp-Pro amide bond. The metal ion, which bonds, inter alia, to the carbonyl oxygen of the proline residue, acts as a fixed charge site, and directs a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues are sequentially cleaved from the C-terminal end, and the terminal aziridinone structure moves one step toward the N-terminus with each C-terminal amino acid residue removed. These observations are the basis of a new method to sequence cyclic peptides. Amino acid residues are observed as sets of three ions, a*(n)PD, b*(n)PD and c*(n)PD where n is the number of amino acid residues in the peptide.

Multistep tandem mass spectrometry for sequencing cyclic peptides in an ion-trap mass spectrometer.

Collisionally activated decomposition (CAD) of a protonated cyclic peptide produces a superposition spectrum consisting of fragments produced following random ring opening of the cyclic peptide to give a set of acylium ions (or isomeric equivalents) of the same m/z. Assignment of the correct sequence is often difficult owing to lack of selectivity in the ring opening. A method is presented that utilizes multiple stages of CAD experiments in an electrospray ion-trap mass spectrometer to sequence cyclic peptides. A primary acylium ion is selected from the primary product-ion spectrum and subjected to several stages of CAD. Amino-acid residues are sequentially removed, one at each stage of the CAD, from the C-terminus, until a b2 ion is reached. Results are presented for seven cyclic peptides, ranging in sizes from four to eight amino-acid residues. This method of sequencing cyclic peptides eliminates ambiguities encountered with other MS/MS approaches. The power of the strategy lies in the capability to execute several stages of CAD upon a precursor ion and its decomposition products, allowing the cyclic peptide to be sequenced in an unambiguous, stepwise manner.

A nomenclature system for labeling cyclic peptide fragments.

A nomenclature system for labeling fragment ions of cyclic peptides is proposed. A fragment ion is labeled with a four-part descriptor for the general formula xnJZ, where x is the designation for the ion (e.g., lower case a or b, as are used for peptide fragments) and n is the number of amino acid residues in the ion. A b ion is the usual acylium ion or isomeric equivalent consisting of n amino-acid residues, and this ion may lose carbon monoxide to form an a ion. The subscripts J and Z are the one-letter (upper-case) codes for the two amino-acid residues connecting the backbone amide or ester bond, J-Z, that can be viewed as broken to form the decomposing linear ion. The symbol J is for the N-terminal amino-acid residue, and Z is that for the C-terminal amino-acid residue that result from the bond cleavage. The nomenclature system is applicable to a wide range of cyclic peptides, including depsipeptides, cyclic peptides containing modified or unusual amino acids, cyclic peptides bearing a linear peptide moiety, and cyclic peptides that are introduced into the gas phase as metal-ion cationized species.

Novel sodium binding properties of some cyclopentapeptide endothelin A selective receptor antagonists: electrospray and fast-atom-bombardment mass spectrometric studies.

Electrospray ionization and fast atom bombardment mass spectrometric methods reveal novel interactions of endothelin A selective receptor antagonists, cyclo(D-Trp-D-Asp-Pro-D-Val-Leu), cyclo(D-Trp-D-Glu-Ala-D-allo-Ile-Leu) and cyclo(D-Trp-D-Asp-Pro-D-Ile-Leu) with sodium ions. The peptides have very high intrinsic affinities for sodium ions, and form multiple sodium adducts and sandwich structures: [M + Na]+, [M + 2Na - H]+, [M + 3Na - 2H]+, [M + 4Na - 3H]+, [M + 5Na - 4H]+, [2M + Na]+, [2M + 2Na - H]+, [2M + 3Na - 2H]+, [2M + 4Na - 3H]+, [2M + 5Na - 4H]+, [2M + 6Na - 5H]+, and [2M + 7Na - 6H]+. The three cyclic peptides exhibit similar sodium binding stoichiometries despite differences in their amino acids. The observed sodium binding properties may have implications in understanding their protective effects against ischemia-induced acute renal failure. Those cyclic peptides that offer protection may be those that have high affinities for multiple sodium ions.

Effects of cations and charge types on the metastable decay rates of oligosaccharides.

Metastable decay rates of alpha-cyclodextrin and maltohexaose coordinated to proton and alkali metal ions were determined from ions produced by liquid secondary ion mass spectrometry in an external source Fourier transform mass spectrometry instrument. For both oligosaccharide compounds the decay rates of the protonated species are faster than any alkali metal coordinated species. Decay rates of the metal cationized species decrease in the order Li+, Na+, K+, and Cs+. The anion of alpha-cyclodextrin has the slowest measurable decomposition rate. The relationships between cation affinities and rates are explored.

Comparative study of the effect of ferrocyanide and EDTA on the production of ethyl alcohol from molasses by Saccharomyces cerevisiae.

The effects of potassium ferrocyanide and EDTA on ethyl alcohol production from molasses by Saccharomyces cerevisiae were investigated on simulated batch pilotplant-scale conditions for alcoholic fermentation of molasses. Ethyl alcohol production was more sensitive to ferrocyanide than to EDTA. When ferrocyanide was introduced into the cultures at the time of inoculation, there was stimulation of ethyl alcohol production, with 261 ppm ferrocyanide producing the maximum effect, which was 3.0% more than in control cultures. When added during the propagation of the yeast, ferrocyanide depressed ethyl alcohol production by 4.0% maximum whereas EDTA stimulated ethyl alcohol production by 2.0%. Addition of ferrocyanide during the fermentation stage produced no significant effect on alcohol production, whereas over a wide range of EDTA concentration there was a steady increase in alcohol yield.