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P Juhasz

Publications and source records attributed to P Juhasz.

16 recordsLinked to original sources
PubMed2000

The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer.

A new matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometer is described for sequencing peptides. This instrument combines the advantages of high sensitivity for peptide analysis associated with MALDI and comprehensive fragmentation information provided by high-energy collision-induced dissociation (CID). Unlike the postsource decay technique that is widely used with MALDI-TOF instruments and typically combines as many as 10 separate spectra of different mass regions, this instrument allows complete fragment ion spectra to be obtained in a single acquisition at a fixed reflectron voltage. To achieve optimum resolution and focusing over the whole mass range, it may be desirable to acquire and combine three separate sections. Different combinations of MALDI matrix and collision gas determine the amount of internal energy deposited by the MALDI process and the CID process, which provide control over the extent and nature of the fragment ions observed. Examples of peptide sequencing are presented that identify sequence-dependent features and demonstrate the value of modifying the ionization and collision conditions to optimize the spectral information.

Arginine↗
PubMed1997

Accurate mass measurements using MALDI-TOF with delayed extraction.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is now an essential tool in biopolymer analysis. Sensitivity and mass range are unsurpassed, but mass measurement accuracy and resolution have been limited. With delayed extraction and a reflecting analyzer, mass measurements using MALDI-TOF can be made with an accuracy of a few parts per million (ppm). It is possible to distinguish Lys from Gln in peptides, and to determine the elemental composition of smaller molecules (mass 100-500). In database searching strategies, a smaller mass window, resulting from an increase in mass accuracy, greatly decreases the number of possible candidates. Mass measurement accuracy with errors less than 5 ppm is demonstrated on a mixture of 12 peptides ranging in mass from ca. 900 to 3700 Da. Mass measurements on 13 peaks in an unseparated tryptic digest of myoglobin gave results with an overall average error less than 3.5 ppm, with a maximum error of 7 ppm.

Adrenocorticotropic Hormone↗
PubMed1996

Sequencing oligonucleotides by exonuclease digestion and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A protocol was developed for sequencing oligonucleotides by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Oligonucleotides were partially hydrolyzed in separate time-course digestions with 3' --> 5' and 5' --> 3' acting phosphodiesterases in a MALDI-compatible buffer or in the MALDI matrix itself. The partial digests were analyzed by MALDI-TOF mass spectrometry employing an instrument equipped with delayed ion extraction and the sequence was inferred from the mass differences between adjacent peaks. Resolution, mass accuracy, and sensitivity were considerably enhanced with delayed extraction, in comparison with the standard MALDI technique. Much longer lengths of DNA can be unambiguously sequenced with delayed extraction MALDI compared with standard MALDI-MS.

Base Sequence↗
PubMed1996

DNA sequencing by delayed extraction-matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a delayed ion extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40- and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.

Base Sequence↗
PubMed1996

Applications of delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry to oligonucleotide analysis.

A delayed ion extraction technique is shown to dramatically improve mass resolution and the overall quality of matrix-assisted laser desorption ionization (MALDI) mass spectra of oligonucleotides. Isotope limited mass resolution was obtained on samples up to 10-kDa molecular mass in linear mode, and as high as 7500 mass resolution (defined at half peak height) was observed in reflector mode. This performance is as good as that achieved to date for peptides and proteins. Applications included the detection of oxidized byproducts of phosphorothioate DNA and separation of components differing only by 15 Da at 9.5-kDa molecular mass. In addition to single components, complex mixtures could also be analyzed at greatly improved performance over conventional MALDI. An example is shown for sequence verification of an oligonucleotide of 31 bases in length by analyzing the failure products. Mass accuracy was adequate to verify sequences of oligodeoxyribonucleotides up to 9500-Da molecular mass. Fast fragmentation taking place between the ionizing pulse and the extraction pulse is demonstrated to be a sequencing tool for small oligonucleotides. By proper selection of matrix material, wavelength, and irradiance, fast fragmentation can be promoted efficiently. Fragment ions tend to form from cleavage of phosphodiester bonds, as previously observed in infrared MALDI.

Base Sequence↗
PubMed1996

Novel neuropeptide Y processing in human cerebrospinal fluid from depressed patients.

In vitro processing of neuropeptide Y (NPY) in cerebrospinal fluid (CSF) of patients with depression was monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Single peptide bonds in NPY were cleaved to yield N- or C-terminal fragments. Multiple cleavage to form internal peptides was unimportant. Degradation rates varied between individuals, whereas the product distributions were fairly constant. Other peptides did not evidence such proteolysis. MALDI-TOF MS will facilitate extensive investigations of NPY processing that could provide the basis for clinical assays and illuminate the pathophysiology related to depression.

Adult↗
PubMed1995

Utility of non-covalent complexes in the matrix-assisted laser desorption ionization mass spectrometry of heparin-derived oligosaccharides.

Molecular weights of heparin-derived oligosaccharides ranging from disaccharides to hexadecasaccharides have been determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. While these compounds ionize poorly or not at all when used as such, a strong signal can be obtained of their ionic complexes formed with a basic peptide or protein. The molecular weight of the sulfated oligosaccharide is determined by subtracting the mass of the basic component from that of the complex. Optimization of the experimental conditions resulted in sub-picomole sensitivity, in the elimination of sulfate loss and of the interference from attachment of inorganic cations. Synthetic peptides (Arg-Gly)10 and (Arg-Gly)15 were specifically designed as complexing agents for synthetic and natural heparin fragments up to decasaccharides. Accurate molecular weight determination on chemically homogeneous oligosaccharides (+/- 0.05%) unambiguously identified the number of saccharide units, and the number of O,N-sulfate and N-acetyl groups. For oligosaccharides larger than decasaccharides, a small basic protein, angiogenin (M(r) = 14,120), was used to form the complex (an inhomogeneous hexadecasaccharide fraction was the largest available for this study). For inhomogeneous samples larger than decasaccharides, the mass accuracy is lower (+/- 0.2-0.3%) but still suffices to determine the number of saccharide units present and to estimate the number of sulfate groups, except it is no longer possible to differentiate one sulfate from two N-acetyl groups (delta = 4 Da). However, taking into account known regularities of sulfation and acetylation, the specificity of heparin lyases and chemical degradation steps, the method promises to contribute significantly to the determination of the primary structure of heparin and other sulfated glycosaminoglycans.

Amino Acid Sequence↗
PubMed1994

Mass spectrometric molecular-weight determination of highly acidic compounds of biological significance via their complexes with basic polypeptides.

Highly acidic compounds that are difficult to ionize by matrix-assisted laser desorption ionization give excellent spectra when mixed with a basic peptide or protein to form a noncovalent complex. This phenomenon makes it possible to determine the molecular weights of polysulfated, -sulfonated, and -phosphorylated biomolecules such as cysteic acid-containing peptides, oligonucleotides, heparin-derived oligosaccharides, and suramin (a drug containing two trisulfonated naphthalene moieties). Peptides and small proteins rich in arginine were used as the basic components. The extent of complex formation correlates with the number of phosphate and sulfate groups in the acidic component and with the number of arginines in the basic component. Neither the acidic amino acid residue aspartic and glutamic acid nor the basic lysine and histidine contribute to complex formation. For oligonucleotides, histone H4 was found to be the best complexing agent investigated. The analytical utility of the complex formation is demonstrated by the molecular-mass determination of acidic compounds from 500 to 6000 Da at the picomole or sub-picomole level with an accuracy of +/- 0.1% or better and by the absence of alkali cation adducts.

Amino Acid Sequence↗
PubMed1993

Generation of large radical ions from oligometallocenes by matrix-assisted laser desorption ionization.

Non-polar polymers containing ferrocene, ferrocenylnaphthalene, and ruthenocenylnaphthalene groups in their repeating units were studied by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry. Sample preparation for these polymers utilized tetrahydrofuran as solvent and several new matrices such as dithranol, 9-nitroanthracene, and quinizarin (all of these are anthracene derivatives). By comparing the mass spectra of oligometallocenes recorded with different matrices at different wavelengths (337 nm and 2.94 microns) and laser desorption ionization mass spectra recorded without matrix, it could be verified that the major analyte-related peaks in the MALDI mass spectra corresponded to the radical molecular ions. Radical ions have not been seen in the MALDI mass spectra of biopolymers such as proteins, peptides, and carbohydrates. Radical formation was demonstrated for samples in the mass range (1 kDa-13 kDa). Even in the presence of potential cationization sites such as methyl ester groups in the repeating units of some polyferrocene samples, MALDI mass spectra were dominated by radical ions of the analyte. Two possible mechanisms for radical formation in MALDI are discussed. Comparison of results with different matrices suggested that the distribution of masses observed in the mass spectra and characterized by the polydispersity index was independent of the matrix, but significant differences (5%) in the average molecular weights of the mass distributions were found.

Chemical Phenomena↗
PubMed1982

Influence of 3-methylcholanthrene on liver nucleolar and nucleoplasmic activities of protein kinases and RNA polymerases.

The experiments were designed to investigate some details of the action of 3-methylcholanthrene (3-MC) on the regulation of transcription. After a single intraperitoneal dose of 3-MC a significant increase in the activities of both nucleolar and nucleoplasmic protein kinases in hepatic cells of young rats was found. The maximal stimulation took place 24 hr after the administration of 3-MC and the extent of activation was much greater in the nucleolar fraction. There is a significant elevation of the activities of both functional forms, free and template-engaged, of RNA polymerase A 24 hr after a single injection of 3-MC. Free and engaged forms of extranucleolar RNA polymerase B show a different behaviour: after 24 hr of 3-MC administration the engaged form is markedly enhanced while the activity of the free enzyme shows a significant decrease. The more moderate increase in total RNA polymerase B activity is obviously preceded by a transfer of the enzyme from 'free' to 'engaged' form. Since the enhancement of protein kinase activities was accompanied by the stimulation of nuclear RNA polymerases we suggest that both kinds of enzymes are involved in an epigenetic mechanism of the inducing action of 3-MC on cytochrome P1-450.

Animals↗
PubMed1980

Hepatic nuclear free and engaged RNA-polymerases in young rats pretreated with 3-methylcholanthrene.

In 3-methylcholanthrene (3-MC)-pretreated young rats the free and engaged RNA polymerases were investigated in detail both in the nucleolar and nucleoplasmic fractions. Both functional forms of the nucleolar RNA polymerase (A) show a great stimulation. The maximum increase of the free form up to 235% compared with controls is obtained 24 h after 3-MC administration. The engaged fraction reaches its maximum later. The extranucleolar RNA polymerase (B) in its engaged form shows the maximum effect (160%) after 24 h, whereas the free form is at first diminished and later on increases steadily. The activation of RNA polymerase A and the interconversion process between the free and engaged form of RNA polymerase B seem to play a significant role in the 3-MC-mediated induction.

Amanitins↗
PubMedDate not supplied

Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: implications for proteome mapping.

Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of alpha- and beta-chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa alpha-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.

Acrylic Resins↗
PubMedDate not supplied

Results and prognosis following surgery for malignant tumors of the skull base.

A series of 155 malignant craniofacial tumors referred to a specialized craniofacial center for surgical treatment are presented. One quarter of these were primary and three quarters recurrent. The statistics of long-term follow-up are presented, and it is concluded that this type of surgery for these difficult problems is worthwhile. The increasing experience gained from operating on and following up these patients is presented.

Adolescent↗