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D B Kassel

Publications and source records attributed to D B Kassel.

27 records · Page 2Linked to original sources
PubMed1990

Differentiation of hydroxyproline isomers and isobars in peptides by tandem mass spectrometry.

The isomeric 3- and 4-hydroxyprolines are isobaric with the isomers leucine and isoleucine, and all four have, therefore, the same "residue mass" of 113. Secondary fragmentation processes were found that differentiate the hydroxyproline isomers from each other and from the leucines. Variants of synthetic bradykinin containing one or two hydroxyproline moieties were prepared by using manual Edman degradation and/or enzymatic methods. The tandem mass spectra of these peptides were recorded. The C-terminal wn fragment ions allow the differentiation of 4-hydroxyproline from the 3-isomer and isoleucine, while the N-terminal an ions containing 4-hydroxyproline undergo H2O elimination to differentiate this amino acid from the 3-isomer and leucine. Lys-C digestion of a mussel adhesive protein produced a set of decapeptides varying in the degree of hydroxylation of proline and tyrosine. Heterogeneity with respect to 3-hydroxyproline and 4-hydroxyproline at a certain position in these peptides was assessed by tandem mass spectrometry based on the wn ion series in the CID spectra of these Lys-C peptides. Some N-terminal ions further allow for the differentiation of these two isomeric species.

Hydroxyproline↗
PubMed1989

Determination of [U-13C]glucose turnover into various metabolite pools for the differential diagnosis of lactic acidemias.

Stable-isotope tracer experiments performed in vitro are evaluated for their utility in differentiating between pyruvate dehydrogenase and cytochrome oxidase deficiencies, two of several enzyme defects commonly associated with the lactic acidemias. Fibroblasts of enzyme-deficient individuals and of age-matched controls are grown in medium containing [U-13C]glucose. Direct analysis of cells and conditioned culture medium provides only minor differences in the organic acid/amino acid GC-MS profiles, making differentiation of enzyme defects difficult by this method. However, differences have been found in the glucose turnover into various cell metabolites, making differentiation of these two enzyme defects possible. The cellular pool of glutamic acid experiences 13C-enrichment in both the control and cytochrome oxidase deficient lines, but not in the pyruvate dehydrogenase-deficient line. The cellular pool of an unknown, possibly an aminopentose sugar, on the other hand, experiences 13C-enrichment in the pyruvate dehydrogenase and control lines, but not in the cytochrome oxidase line. These observations, as well as other differences in the extent of enrichment into various metabolite pools, suggest that this stable-isotope approach, in vitro, is feasible and may allow these two enzyme defects to be differentiated in a definitive manner. Such stable-isotope experiments are easy to carry out with cultured cells and are inexpensive. Applications of the technique to other genetic disorders might be appropriate.

Amino Acids↗
PubMed1989

Mass spectrometric analysis of cardiac glycosides by the desorption/ionization technique potassium ion ionization of desorbed species.

The analysis of cardiac glycosides by the desorption/ionization (D/I) mass spectrometric technique potassium ion ionization of desorbed species (K+IDS) is presented. K+IDS mass spectra of digitonin, digoxin, digoxigenin, digitoxin and ouabain are discussed to demonstrate the capabilities of this D/I method. The K+IDS analysis consists of two steps: thermal desorption of neutral molecules representative of the analyte, followed by gas-phase addition of K+ ions to these species. Structural and molecular weight information of the cardiac glycosides is obtained with the K+IDS technique. The most intense peak in the K+IDS mass spectrum of an analyte, M, is frequently the [M]K+ ion. Interpretation of the K+IDS mass spectra is simple, since one thermal degradation mechanism dominates. This mechanism is a 1,2-elimination process. A variation of the original K+IDS technique, performed by changing the ionizing metal from K+ to Na+ (i.e. Na+IDS), is presented for the analysis of digoxin. The Na+IDS mass spectrum of digoxin contains more structural information than the K+IDS mass spectrum of that compound. This may lead to a means of controlling the types of information obtainable with this D/I technique by varying the cation that is thermionically generated. K+IDS analyses can be performed rapidly, no sample derivatization is necessary, no matrix is required and little instrument modification is necessary.

Cardiac Glycosides↗
PubMed1988

Potassium ion ionization of desorbed species (K+IDS): a rapid method for the screening of urine for organic acidemias.

A new desorption/ionization mass spectrometric technique, K+ ionization of desorbed species (K+IDS), is evaluated as a rapid method for differentiating various organic acidemias, conditions in which excessive levels of organic acid metabolites are present in plasma or urine as a result of some inborn error of metabolism. This method requires no derivatization of the isolated organic acids, unlike that required for gas chromatographic and gas chromatographic/mass spectrometric analyses. 'Batch' mass spectrometric analysis is achieved by deposition of the complex organic acid mixture (from urine) onto a K+IDS probe. Rapid heating results in the emission of alkali ions (Na+ or K+) from a thermionic emitter and the intact desorption of analyte. Subsequent gas-phase addition produces a mass spectrum showing alkali ion adducts of the components, providing molecular weight and relative concentration information. This rapid desorption/ionization technique requires no matrix, and analysis times are exceedingly short relative to those required in gas chromatographic/mass spectrometric analyses. Results suggest that differential diagnosis of some of the more commonly occurring organic acidemias (e.g. isovaleric acidemia, maple syrup urine disease, etc.) may be made.

Amino Acid Metabolism, Inborn Errors↗
PubMed1986

Urinary metabolites of L-threonine in type 1 diabetes determined by combined gas chromatography/chemical ionization mass spectrometry.

Metabolic profiling of urinary organic acids from patients with juvenile-onset (Type 1) diabetes mellitus have revealed significantly elevated levels of 2-hydroxybutyric and 4-deoxythreonic acids. To test the hypothesis that these metabolites, as well as 4-deoxyerythronic acid, are derived from L-threonine, stable isotope-labeled threonine was infused into an insulin-deficient dog and the incorporation of 13C into these metabolites was monitored by gas chromatography/mass spectrometry. Electron ionization was relatively insensitive, but positive chemical ionization with ammonia as the reactant gas gave both protonated molecules and [M + NH4]+ ions, which could be analysed by selected ion monitoring. The isotope-labeled species of 2-hydroxybutyric, 4-deoxyerythronic and 4-deoxythreonic acids were observed, but 13C was not incorporated into other organic acids. Thus, it is proposed that L-threonine is a precursor of these metabolites.

Animals↗
PubMedDate not supplied

Realities of high-throughput liquid chromatography/mass spectrometry purification of large combinatorial libraries: a report on overall sample throughput using parallel purification.

We report on the development of a validated, streamlined high-throughput process for the purification of parallel-synthesis-derived combinatorial libraries. The steps involved in this library purification process include dissolution of dry films of crude synthetic material, dual-column LC/MS purification, dual-column postpurification analysis, quantitation, reformatting, and submission of pure compounds for registration. Although the purification and postpurification analysis times decreased essentially linearly as a function of the number of HPLC columns employed, it was not possible to decrease the total purification process time linearly as a function of the number of columns employed in the system. This was due primarily to the fact that numerous steps in the total purification process are independent of sample analysis and purification (e.g., evaporation, reconstitution, and reformatting, etc.). Additionally, experiments were also performed to assess whether separate gradient pumps were necessary for each channel of this two-channel LC/MS or if acceptable results could be reliably obtained by splitting the flow from one set of gradient pumps between two HPLC columns. On the basis of the parallel, two-column LC/MS system employed in this work, throughput estimates were extrapolated to more massively parallel systems (e.g., four-channel LC/MS).

Amino Acids↗
PubMedDate not supplied

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. When the samples were compared by ion spray ionization mass spectrometry, the molecular mass of one sample was 200 daltons higher than anticipated. Careful analysis of the sample, including atomic emission spectrometry, revealed that a mercury atom was associated with the polypeptide. The mercury presumably resulted from a deprotection step in the synthesis, involving the removal of an acetamidomethyl group from cysteine.

Alkylation↗