Pál Tamás Szabó

Identification of ethyl pyruvate and dihydrocinchonidine adducts by electrospray ionization mass spectrometry

Several ethyl pyruvate and dihydrocinchonidine adducts, formed by non-covalent interactions with alkali cations, have been identified for the first time using electrospray ionization mass and tandem mass spectrometry. This type of adduct may have an important role in asymmetric reactions of pyruvates in the presence of cinchonas.

IDENTIFICATION OF OXYTOCIN AND VASOPRESSIN FROM NEUROHYPOPHYSEAL CELL CULTURE

Our observation that dispersed cultures of neurohypophysis obtained from adult rats are capable of synthesizing and releasing oxytocin and vasopressin is unexpected, because in whole animals these hormones are known only to be stored, not to be produced in the posterior lobe of the pituitary. The hormone content of cell culture medium was elevated from 0 to 129 +/- 14 pg/mg protein for oxytocin and from 0 to 42 +/- 4 pg/mg protein for vasopressin during two weeks as determined by specific radioimmunoassay. By molecular mass and structure determination (tandem mass spectrometry) we have proved that the supernatant of the cell cultures contains not only immunologically but mass spectrometrically identified neurohypophyseal hormones.

Capillary chromatography/microelectrospray mass spectrometry used for the identification of putative cyclin-dependent kinase inhibitory protein in medicago

A home-built capillary chromatography/microelectrospray system was used for peptide mass mapping of a putative cyclin-dependent kinase inhibitor protein with molecular mass of 8.5 kDa. The masses of identified tryptic fragments were then input to a protein database search routine. Daughter ion scans were done only on the most abundant tryptic fragments. On the basis of protein database search results and tandem mass spectrometric measurements the bioactive protein was identified as ubiquitin.

Characterization of the gene for factor C, an extracellular signal protein involved in morphological differentiation of Streptomyces griseus.

The gene encoding factor C (facC), an extracellular signal protein involved in cellular differentiation, was cloned from Streptomyces griseus 45H, and the complete nucleotide sequence was determined. The deduced amino acid sequence was confirmed by HPLC/electrospray ionization-mass spectrometry analysis. The full-length protein consists of 324 amino acids and has a predicted molecular mass of 34,523 Da. The mature extracellular 286 amino acid protein (31,038 Da) is probably produced by cleaving off a 38 amino acid secretion signal sequence. Southern hybridization detected facC in several other Streptomyces strains, but database searches failed to identify a protein with significant homology to factor C. Expression of facC from a low-copy-number vector in S. griseus 52-1 resulted in a phenotypic effect similar to that given by exogenously added factor C protein.

Identification of factor C protein from Streptomyces griseus by microelectrospray mass spectrometry.

Factor C, an extracellular signal protein of cellular differentiation, was studied and significant homology was found to several zinc finger-type regulatory proteins. The complete amino acid sequence, deduced from the gene, that encodes the protein, did not support the hypothesis that this protein might be a zinc finger-type regulatory protein. However, a theoretical single nucleotide insertion in the gene can result in another similarly sized protein containing about 20 His residues, which would be responsible for the high zinc affinity of factor C. The protein sample was reduced, alkylated and then in-gel digested with trypsin. The peptide fragments were then separated by capillary chromatography and identified by microelectrospray mass spectrometry. Peaks of higher intensity were sequenced by tandem mass spectrometry. The identified peptide fragments and the measured molecular mass of factor C protein also confirmed the original sequence of protein, as there was no shift in the open reading frame.

Fragmentation of cyclobutane derivatives upon electron impact: transformation pathways elucidated by mass spectrometric methods and semiempirical quantum chemical calculations

The fragmentation behaviour of cyclobutane derivatives (carboxylic acids, esters, cyclobutanediols, silyl ethers of the hydroxy compounds) was first studied by the GC/MS method under electron impact (EI) ionization. Then, in order to establish the sequence of transformations, MS/MS, and in situ deuteration as well as accurate mass measurements, were performed. Analysis of these measurements revealed characteristic transformation pathways: (i) the substituents were easily cleaved, (ii) in most cases the cyclobutane ring was opened with the rupture of the C–C bonds opposite to each other, (iii) upon ring opening, the most substituted ring C–C bond was cleaved first and preferentially (iv) primary fragmentation was followed by secondary reactions including further cleavage of the ions as well as rearrangement reactions. For cyclobutanediols and their silyl ethers symmetric ring scission was observed providing only one primary fragment with m/z half that of the molecular weight. This type of fragmentation was consistent with semiempirical quantum chemical calculation.

Electron Impact Mass Spectrometry of 2-Aryl-thiazolidine-4-carboxylic Acids

The mass spectrometric behaviour of 11 2-aryl-thiazolidine-4-carboxylic acids has been studied under electron impact conditions. On the basis of our high- and low-resolution measurements, deuterium labelling experiments and collision-induced dissociation studies the main fragmentation pathways have been established. In the high resolution spectrum of 2-phenyl-thiazolidine-4-carboxylic acid several peaks were found to be doublets. Some of the chemical formulae were unexpected. With the help of tandem mass spectrometry the formation of these ions was explained.