Fast atom bombardment combined with tandem mass spectrometry for determining structures of small oligonucleotides.

A study of small (n = 3 to 6) oligonucleotide and the metastable and collisionally activated decompositions of their (M-H)- species desorbed by using fast atom bombardment (FAB) is reported. Data were obtained for both ribo- and 2'-deoxyribotrinucleotides and for 2'-deoxyribotetra-, penta-, and hexanucleotides. The favored metastable decompositions of all of the oligonucleotides studied are eliminations of neutral CONH and loss of BH, where B is the base moiety. The BH elimination, however, provides little sequence information in the higher oligonucleotides and the process is more indicative of the different bases present in the oligomer. The chemistry observed upon collisional activation changes as one goes from trinucleotides to hexanucleotides. The formation of sequence ions is more facile for processes involving the 3' terminus, allowing the sequence to be determined. As one goes to the higher oligonucleotides, however, several different competitive fragmentation processes become as facile as or more facile than the reactions giving the sequence ions. This hinders proper ion assignments and makes sequence determination difficult.

Enzymic synthesis of caffeoylglucaric Acid from chlorogenic Acid and glucaric Acid by a protein preparation from tomato cotyledons.

The phenylpropane metabolism of tomato (Lycopersicon esculentum Mill) cotyledons was investigated. The HPLC analysis revealed two hydroxycinnamic-acid conjugates as major components, identified as chlorogenic acid (5-O-caffeoylquinic acid) and caffeoylglucaric acid (2-O- or 5-O-caffeoyl-glucaric acid). Quantitative analyses indicated a precursor-product relationship between the chlorogenic and caffeoylglucaric acids. Protein preparations from tomato cotyledons were found to catalyze the formation of caffeoylglucaric acid with chlorogenic acid as acyl donor and free glucaric acid as acceptor molecule. This enzyme activity, possibly to be classified as hydroxycinnamoylquinic acid:glucaric acid hydroxycinnamoyltransferase, acts together with hydroxycinnamoyl-CoA: quinic acid hydroxycinnamoyltransferase.

Fast atom bombardment combined with tandem mass spectrometry for the study of dinucleotides.

A study of mono- and dinucleotides by utilizing negative ion fast atom bombardment (FAB), metastable decomposition of (M-H)- species, and collisionally activated decomposition (CAD) of (M-H)- species is reported. Data were obtained for several complete series containing the standard nucleosides (guanosine, adenosine, cytidine, thymidine, and uridine): the 3'- and 5'-monophosphate mononucleotide series for both ribo- and 2'-deoxyribomononucleotides, all possible combinations for the 3'(-)----5'-ribodinucleotides, and all possible combinations of the 3'(-)----5',2'-deoxyribodinucleotides. The metastable and CAD spectra provide more information than the FAB mass spectra. The (M-H)- ions of all dinucleotides decompose either as metastable ions or upon collisional activation to eliminate BH (B = base) preferentially from the 3'- rather than the 5'-terminus. Isomeric dinucleotides can be distinguished on the basis of this fragmentation. To establish the identity of the base at the 5'-terminus, collisional activation is preferred. By comparing relative abundances of BH elimination observed, the inherent basicities of the nucleoside base anions can be inferred to be C- greater than A-, T-, greater than G-.

Structures of the major carbohydrates of natural human interleukin-2.

Purified human interleukin-2 secreted by peripheral blood lymphocytes from healthy donors was found to exist in several forms. These forms were (partially) resolved by reversed-phase high-performance liquid chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Two major polypeptide species (interleukin-2 N1 and N2, 16.5 kDa) were shown to be glycosylated on the basis of [3H]galactose/[3H]glucosamine incorporation and determination of amino sugars after acid hydrolysis. A third component (interleukin-2 M, 14.5 kDa) represents a nonglycosylated form. The amino acid composition and the NH2-terminal sequence of both forms are consistent with the data deduced from the cDNA coding for interleukin-2 after removal of a leader peptide of 20 amino acids. Carbohydrates are O-linked to the IL-2 protein via threonine-3 of the polypeptide chain. The oligosaccharides were released by reductive beta-elimination and were purified by gel filtration and high-performance liquid chromatography. Applying methylation analysis, exoglycosidase digestion and fast atom bombardment mass spectrometry the following major carbohydrate structures were identified: N1, NeuAc(alpha 2-3)Gal(beta 1-3)GalNAc-ol; and N2, NeuAc(alpha 2-3)Gal(beta 1-3)[NeuAc(alpha 2-6)]GalNAc-ol.

Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site.

The synthesis and characterization of an octanucleotide, d(GGsAATTCC), containing the recognition sequence of the EcoRI restriction endonuclease with a phosphorothioate internucleotidic linkage at the cleavage site are described. Two approaches for the synthesis of the RP and SP diastereomers of this octamer by the phosphite method are presented. The first consists of the addition of sulfur instead of H2O to the phosphite at the appropriate position during chain elongation. This method results in a mixture of diastereomers that can be separated by high-performance liquid chromatography after 5'-terminal phosphorylation. The second uses the presynthesized and diastereomerically pure dinucleoside phosphorothioate d[Gp(S)A] for the addition to the growing oligonucleotide chain as a block. The products are characterized by digestion with nuclease P1, fast atom bombardment mass spectrometry, 31P NMR spectroscopy, and conversion to d(GGAATTCC) by desulfurization with iodine. Only the RP diastereomers of d(GGsAATTCC) and its 5'-phosphorylated derivative are cleaved by EcoRI endonuclease. The rate of hydrolysis is slower than that of the unmodified octamer. The phosphorothioate octamer will be useful for the determination of the stereochemical course of the EcoRI-catalyzed reaction.

Direct mass spectroscopic method for determination of oxygen isotope position in adenosine 5'-O-(1-thiotriphosphate). Determination of the stereochemical course of the yeast phenylalanyl-tRNA synthetase reaction.

Negative ion fast atom bombardment mass spectrometry has been used to distinguish between (Sp)-adenosine 5'-O-(1-thiotriphosphate) containing either an alpha- nonbridging or an alpha-beta-bridging 18O label. The method does not require any nucleotide derivatization and so avoids the excessive manipulations and purifications necessary to distinguish between the above two species using conventional mass spectroscopy. Furthermore, it is between 50 and 200 times more sensitive than other direct methods based on 31P nuclear magnetic resonance spectroscopy. Routinely, 100 nmol of nucleoside phosphorothioate is ample to establish the 18O isotope position by normal as well as linked scan mass spectrometry. In cases where normal mass spectrometry is considered adequate, 10 nmol of material suffices. This technique should be useful in determining the stereochemical course of enzymatic nucleotidyl transfer and nuclease-catalyzed hydrolysis reactions under conditions of limiting availability of enzyme or substrate. Yeast phenylalanyl-tRNA synthetase was used to prepare the 18O-labeled adenosine 5'-O-(1-thiotriphosphate) species, and this enzyme was concomitantly shown to catalyze adenylyl transfer with inversion of configuration at phosphorus.

New Acylated Cyanogenic Diglycosides from Fruits of Anthemis cairica.

Three new cyanogenic diglycosides of the mandelonitrile series have been isolated from fruits of ANTHEMIS CAIRICA (Compositae) and their structures identified by degradation and spectral methods mainly (1)H-NMR, (13)C-NMR, FAB-MS. Minor compounds are 2-beta-primeverosyloxy-2-phenyl-2S-acetonitrile (epilucumin) and its 4''-p(beta-D-glucopyranosyloxyl-(E)cinnamate; the main compound is the 4''-p(beta-primeverosyloxy)-(E)cinnamate of epilucumin.

Ultrafast sequencing of oligodeoxyribonucleotides by FAB-mass spectrometry.

A fully instrumental method is described for the bidirectional sequencing of oligodeoxyribonucleotides. The method makes use of the negative ion fragmentation patterns of fast atom bombardment mass spectrometry. It is less time consuming than any other sequencing procedure known to date. Since one sequencing run takes as little as one hour, this new method is anticipated to cut down considerably the time required for the controlled synthesis of oligodeoxyribonucleotides of (currently) up to ten nucleotide units in length.

Iso-branched 2- and 3-hydroxy fatty acids as characteristic lipid constituents of some gliding bacteria.

The fatty acids present in the total hydrolysates of several gliding bacteria (Myxococcus fulvus, Stigmatella aurantiaca, Cytophaga johnsonae, Cytophaga sp. strain samoa and Flexibacter elegans) were analyzed by combined gas-liquid chromatography and mass spectrometry. In addition to 13-methyl-tetradecanoic acid, 15-methyl-hexadecanoic acid, hexadecanoic acid, and hexadecenoic acid, 2- and 3-hydroxy fatty acids comprised up to 50% of the total fatty acids. The majority was odd-numbered and iso-branched. Small amounts of even-numbered and unbranched fatty acids were also present. Whereas 2-hydroxy-15-methyl hexadecanoic acid was characteristic for myxobacteria, 2-hydroxy-13-methyl-tetradecanoic acid, 3-hydroxy-13-methyl-tetradecanoic acid, and 3-hydroxy-15-methyl-hexadecanoic acid were dominant in the Cytophaga-Flexibacter group.

Analysis of calf-thymus satellite DNA: evidence for specific methylation of cytosine in C-G sequences.

Digestion of purified calf tymus satellite I (phi = 1.714 g/cm3) with a series of restriction enzymes shows that modification in this satellite occurs preferentially in the sequence C-G. This was also shown to be the case in the other satellites and in bulk chromosomal calf thymus DNA. Cloning of purified satellite I DNA in Escherichia coli makes sites, previously modified, available for cutting with certain restriction enzymes. All these 'new sites' contain the sequence C-G. High-resolution mass spectros-copy establishes that the satellites contain a low concentration of 5-methylcytosine. This infers that methylation which inhibits retriction enzyme cutting must occur preferentially in the sequence C-G. Hybridization of cRNA of cloned satellite I DNA with the satellites III (phi = 1.706 g/cm3) and IV (phi = 1.710 g/cm3) shows that there is no or little sequence homology between these satellites. Digestion of calf thymus satellite I DNA with endoR. EcoRI and subsequent hybridization studies with the fragments shows two EcoRI fragments in addition to the usual 1400-base-pair EcoRI repeat unit.