Biomedical subjects

K F Medzihradszky

Publications and source records attributed to K F Medzihradszky.

At least 19 recordsLinked to original sources

PubMed2004

Characterization of Tetrahymena histone H2B variants and posttranslational populations by electron capture dissociation (ECD) Fourier transform ion cyclotron mass spectrometry (FT-ICR MS).

This work describes the nature and sequence information content of the electron capture dissociation mass spectra for the intact Tetrahymena histone H2B. Two major variants of this protein were present bearing nominal modifications of both +42 and +84 Da. This work describes identification of the nature of these two modifications. For example, using gas-phase selection and isolation of the +42-Da modified species, from a background of two H2B variants each present in six or more posttranslationally modified isoforms, we were able to determine that this +42-Da modification isoform bears trimethylation rather than acetylation. LC-CIDMS analysis was also employed on digested preparations to obtain complementary detail of the nature of site-specific posttranslational modifications. This study establishes that integration of the information from these two datasets provides a comprehensive map of posttranslational occupancy for each particular covalent assemblage selected for structural investigation.

Amino Acid Sequence↗

PubMed2004

O-sulfonation of serine and threonine: mass spectrometric detection and characterization of a new posttranslational modification in diverse proteins throughout the eukaryotes.

Protein sulfonation on serine and threonine residues is described for the first time. This post-translational modification is shown to occur in proteins isolated from organisms representing a broad span of eukaryote evolution, including the invertebrate mollusk Lymnaea stagnalis, the unicellular malaria parasite Plasmodium falciparum, and humans. Detection and structural characterization of this novel post-translational modification was carried out using liquid chromatography coupled to electrospray tandem mass spectrometry on proteins including a neuronal intermediate filament and a myosin light chain from the snail, a cathepsin-C-like enzyme from the parasite, and the cytoplasmic domain of the human orphan receptor tyrosine kinase Ror-2. These findings suggest that sulfonation of serine and threonine may be involved in multiple functions including protein assembly and signal transduction.

Animals↗

PubMed2001

Phosphorylation of native and heme-modified CYP3A4 by protein kinase C: a mass spectrometric characterization of the phosphorylated peptides.

As an initial approach toward the characterization of the phosphorylation of cumene hydroperoxide (CuOOH)-inactivated cytochrome P450 (CYP3A4, the major human liver drug-metabolizing enzyme) and its role in the degradation of the inactivated protein, we have identified one of the major participating cytosolic kinase(s) as rat liver cytosolic protein kinase C (PKC) with the use of specific and general kinase inhibitors. Accordingly, we employed a model phosphorylation system consisting of purified PKC, gamma-S-[(32)P]ATP, and either native or CuOOH-inactivated purified recombinant His(6)-tagged CYP3A4. Lysylendoprotease (Lys)-C digestion of the phosphorylated CuOOH-inactivated CYP3A4(His)(6) followed by HPLC-peptide mapping and mass spectrometric (LC/MS/MS) analyses led to the isolation and the unambiguous identification of two PKC-phosphorylated CYP3A4 peptides: E(258)SRLEDT(p)QK(266) and F(414)LPERFS(p)K(421). Similar analyses of the PKC-phosphorylated native enzyme predominantly yielded E(258)SRLEDT(p)QK(266) as the phosphorylated peptide. Studies are currently in progress to determine whether phosphorylation of any or both of these peptides is required for the Ub-dependent 26S proteasomal degradation of CuOOH-inactivated CYP3A4.

Amino Acid Sequence↗

PubMed2001

Characterization of glycated hemoglobin in diabetic patients: usefulness of electrospray mass spectrometry in monitoring the extent and distribution of glycation.

A combination of chromatographic and mass spectrometric techniques was used to evaluate the extent and distribution of glycation within the glycated hemoglobin (GHb) molecule. Studies on quantification of hemoglobin (Hb) glycation by electrospray ionization mass spectrometry (ES-MS) of intact globins employed specimens from 10 diabetic individuals and five normal controls. Detailed structural analysis of the phenylboronate affinity chromatography/ion-exchange (IE) HPLC-separated sub-populations of GHb was performed on a specimen carrying 13.7% GHb. An efficient protocol for mapping glycation sites within alpha and beta globins was developed, e.g., Glu-C/Asp-N proteolytic digestion followed by LC-ES-MS. Relative site occupancy within discrete components of GHb was evaluated. A correlation between the degree of glycation measured at Hb level (by affinity chromatography) and at globin level (measured by ES-MS) was carried out. The above studies led us to conclude that during the process of phenylboronate chromatography GHb dimers, rather than tetramers, are bound to the affinity resin so a fraction of glycated dimers rather than tetramers is measured. This finding implies that a process of glycation affects a much higher number of native Hb tetramers than was previously contemplated. No glycation sites appear to be missed by phenylboronate affinity chromatography. We have found no evidence of the presence of multiple glycations within a single globin chain. While glycation of both globins within a dimer cannot be excluded, it is unlikely to be a significant phenomenon. According to ES-MS data, an equivalent of about one globin per alphabeta dimer of the affinity chromatography-isolated GHb carried glycation.

Amino Acid Sequence↗

PubMed2001

Matrix-assisted laser desorption/ionization coupled with quadrupole/orthogonal acceleration time-of-flight mass spectrometry for protein discovery, identification, and structural analysis.

The design and operation of a novel UV-MALDI ionization source on a commercial QqoaTOF mass spectrometer (Applied Biosystem/MDS Sciex QSTAR Pulsar) is described. Samples are loaded on a 96-well target plate, the movement of which is under software control and can be readily automated. Unlike conventional high-energy MALDI-TOF, the ions are produced with low energies (5-10 eV) in a region of relatively low vacuum (8 mTorr). Thus, they are cooled by extensive low-energy collisions before selection in the quadrupole mass analyzer (Q1), potentially giving a quasi-continuous ion beam ideally suited to the oaTOF used for mass analysis of the fragment ions, although ion yields from individual laser shots may vary widely. Ion dissociation is induced by collisions with argon in an rf-only quadrupole cell, giving typical low-energy CID spectra for protonated peptide ions. Ions separated in the oaTOF are registered by a four-anode detector and time-to-digital converter and accumulated in "bins" that are 625 ps wide. Peak shapes depend upon the number of ion counts in adjacent bins. As expected, the accuracy of mass measurement is shown to be dependent upon the number of ions recorded for a particular peak. With internal calibration, mass accuracy better than 10 ppm is attainable for peaks that contain sufficient ions to give well-defined Gaussian profiles. By virtue of its high resolution, capability for accurate mass measurements, and sensitivity in the low-femotomole range, this instrument is ideally suited to protein identification for proteomic applications by generation of peptide tags, manual sequence interpretation, identification of modifications such as phosphorylation, and protein structural elucidation. Unlike the multiply charged ions typical of electrospray ionization, the singly charged MALDI-generated peptide ions show a linear dependence of optimal collision energy upon molecular mass, which is advantageous for automated operation. It is shown that the novel pulsing technique of this instrument that increases the sensitivity for precursor ions scans is applicable to the identification of peptides labeled with isotope-coded affinity tags.

Databases, Factual↗

PubMed2001

Protein identification by in-gel digestion, high-performance liquid chromatography, and mass spectrometry: peptide analysis by complementary ionization techniques.

A biologically active protein fraction was isolated from rabbit intestine, purified by one-dimensional SDS-PAGE and stained with Coomassie Brilliant Blue. A predominant band of approximately 110-130 kDa was excised and digested in-gel with trypsin. The resulting peptides were extracted then separated by microbore reversed-phase high-performance liquid chromatography (HPLC). Mass spectrometric data from one HPLC fraction obtained by two different ionization techniques proved to be complementary. Matrix-assisted laser desorption/ionization (MALDI) showed nine peptide masses, which by post source decay analysis and database searching were attributed to two proteins. Nanoflow electrospray analysis performed on a hybrid tandem mass spectrometer of quadrupole-quadrupole-orthogonal acceleration time-of-flight (QqTOF) geometry detected six additional peptide components. On the basis of the additional peptides and superior quality collision-induced dissociation spectra typical of this instrument type, two further proteins were identified. The resolution afforded by the QqTOF instrument permitted charge state determination for the fragment ions while preserving the high detection sensitivity that was essential in obtaining the composition of this mixture of proteins.

Animals↗

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↗

PubMed2000

Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools.

BACKGROUND: Analysis of global changes in gene transcription and translation by systems-based genomics and proteomics approaches provides only indirect information about protein function. In many cases, enzymatic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly determining activities of an entire class of enzymes on a genome-wide scale would be of great utility. RESULTS: We have engineered chemical probes that can be used to broadly track activity of cysteine proteases. The structure of the general cysteine protease inhibitor E-64 was used as a scaffold. Analogs were synthesized by varying the core peptide recognition portion while adding affinity tags (biotin and radio-iodine) at distal sites. The resulting probes containing a P2 leucine residue (DCG-03 and DCG-04) targeted the same broad set of cysteine proteases as E-64 and were used to profile these proteases during the progression of a normal skin cell to a carcinoma. A library of DCG-04 derivatives was constructed in which the leucine residue was replaced with all natural amino acids. This library was used to obtain inhibitor activity profiles for multiple protease targets in crude cellular extracts. Finally, the affinity tag of DCG-04 allowed purification of modified proteases and identification by mass spectrometry. CONCLUSIONS: We have created a simple and flexible method for functionally identifying cysteine proteases while simultaneously tracking their relative activity levels in crude protein mixtures. These probes were used to determine relative activities of multiple proteases throughout a defined model system for cancer progression. Furthermore, information obtained from libraries of affinity probes provides a rapid method for obtaining detailed functional information without the need for prior purification/identification of targets.

Affinity Labels↗

PubMed1999

Spin trapping and protein cross-linking of the lactoperoxidase protein radical.

Lactoperoxidase (LPO) reacts with H(2)O(2) to sequentially give two Compound I intermediates: the first with a ferryl (Fe(IV)=O) species and a porphyrin radical cation, and the second with the same ferryl species and a presumed protein radical. However, little actual evidence is available for the protein radical. We report here that LPO reacts with the spin trap 3,5-dibromo-4-nitroso-benzenesulfonic acid to give a 1:1 protein-bound radical adduct. Furthermore, LPO undergoes the H(2)O(2)-dependent formation of dimeric and trimeric products. Proteolytic digestion and mass spectrometric analysis indicates that the dimer is held together by a dityrosine link between Tyr-289 in each of two LPO molecules. The dimer retains full catalytic activity and reacts to the same extent with the spin trap, indicating that the spin trap reacts with a radical center other than Tyr-289. The monomeric protein recovered from incubations of LPO with H(2)O(2) is fully active but no longer forms dimers when incubated with H(2)O(2), clear evidence that it has also been structurally modified. Myeloperoxidase, a naturally dimeric protein, and eosinophil peroxidase do not undergo H(2)O(2)-dependent oligomerization. Analysis of the interface in the LPO dimers indicates that the same protein surface is involved in LPO dimerization as in the normal formation of myeloperoxidase dimers. Oligomerization of LPO alters its physical properties and may alter its ability to interact with macromolecular substrates.

Animals↗

PubMed1999

Fragmentation and sequencing of cyclic peptides by matrix-assisted laser desorption/ionization post-source decay mass spectrometry.

A series of synthetic cyclic decapeptides and other smaller cyclic peptides were analyzed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The investigated compounds were cyclized in a head-to-tail manner and contained non-proteinaceous amino acids, such as D-phenylalanine, D,L-4-carboxyphenylalanine, epsilon-aminocaproic acid, and gamma-aminobutyric acid, and were synthesized in a program to develop inhibitors of pp60(c-src) (Src), a tyrosine kinase that is involved in signal transduction and growth regulation. Post-source decay (PSD) spectra of the cyclic peptides featured abundant sequence ions. Two preferential ring opening reactions were detected resulting in linear fragment ions with an N-terminus of proline and a C-terminus of glutamic acid, respectively. MALDI-PSD spectra even permitted de novo sequencing of some cyclic peptides. Systematic studies on cyclic peptides using this method of fragmentation have not been reported to date. This work presents an easy mass spectrometric method, MALDI-PSD, for the characterization and identification of cyclic peptides.

Esters↗

PubMed1999

Study on the synthesis and characterization of peptides containing phosphorylated tyrosine.

Phosphorylation and dephosphorylation are key events in receptor-mediated and post-receptor-mediated signal transduction. Synthetic phosphopeptides have been shown to have dramatic agonist or antagonist effects in several of these signaling pathways. For its 1997 study, the Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group assessed the ability of member laboratories to synthesize phosphotyrosine peptides. Participating laboratories were requested to synthesize and submit the following crude peptide, H-Glu-Asp-Tyr-Glu-Tyr(PO3H2)-Thr-Ala-Arg-Phe-NH2, for evaluation by amino acid analysis, sequence analysis, RP-HPLC, MALDI-TOF and ESI mass spectrometry. Prior to analysis of submitted peptides from ABRF members, the Peptide Synthesis Research Group synthesized and characterized the nonphosphorylated form of the peptide, the doubly phosphorylated form and the peptides singly phosphorylated on either the first or the second tyrosine. These peptide standards were separated easily by HPLC and capillary electrophoresis and the phosphotyrosine was detected readily by Edman degradation sequence analysis. No differences were seen by amino acid analysis and the expected masses were observed by mass spectrometry. The two singly phosphorylated peptides were easily distinguished by MALDI-PSD. Analysis of the peptides submitted from member facilities revealed that all but four of the 33 samples contained the correct product as determined by HPLC and mass spectrometry. HPLC analysis indicated that 20 of the 33 submitted samples contained greater than 75% correct product, five contained less than 50% correct product and four did not contain any correct product. By ESI/MS, an additional singly charged ion at m/z 535.5 was detected in five of the 33 submitted samples; this ion was subsequently shown to represent Ac-TARF-NH2. No correlation was found to exist between coupling time and percentage correct product; however, a correlation may exist between a greater percentage of correct product and the use of non-protected phosphotyrosine.

Biochemistry↗

PubMed1998

Human low-molecular-weight salivary mucin expresses the sialyl lewisx determinant and has L-selectin ligand activity.

Previously we showed that the low-molecular-weight mucin (MG2, encoded by MUC7), a major component of human submandibular/sublingual saliva, is a bacterial receptor that coats the tooth surface. Here we tested the hypothesis that the structure of its carbohydrate residues contains important information about its function. Purified MG2 (Mr 120 000) was digested with trypsin, and the resulting Mr 90 000 fragment, which carried primarily O-linked oligosaccharides, was subjected to reductive beta-elimination. The released oligosaccharides were characterized by using nuclear magnetic resonance spectroscopy and mass spectrometry. Of the 41 different structures we detected, the most prominent included NeuAcalpha2-->3Galbeta1-->3GalNAc-ol (sialyl-T antigen), Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->6(Galbeta1 -->3)GalNAc-ol [type 2 core with Lewisx (Lex) determinant], and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6(Galbeta1--> 3) GalNAc-ol [type 2 core with sialyl Lex (sLex) determinant]. We also detected di-, tri-, and pentasaccharides with one sulfate group. Lex, sLex, and related sulfated structures are ligands for selectins, adhesion molecules that mediate leukocyte trafficking. Therefore, we investigated whether MG2 was a selectin ligand. In an enzyme-linked immunosorbent assay, L-selectin chimeras interacted with immobilized MG2 in a Ca2+-dependent manner. L-Selectin chimeras also bound to MG2 immobilized on nitrocellulose. Together, these results suggest that the saccharides that MG2 carries could specify some of its important functions, which may include mediating leukocyte interactions in the oral cavity.

Carbohydrate Sequence↗

PubMed1998

Heme oxygenase active-site residues identified by heme-protein cross-linking during reduction of CBrCl3.

The reduction of CBrCl3 by the heme-heme oxygenase complex forms dissociable and covalently bound heme products. No such products are formed with mesoheme in which the heme vinyl substituents are replaced by ethyl groups. The dissociable heme products are chromatographically similar but not identical to those obtained in the analogous reaction with myoglobin. Tryptic digestion of the heme-protein adduct and Edman sequencing and mass spectrometric analysis of the heme-linked peptide identify His-25, the proximal iron ligand, as the alkylated residue. Reaction of CBrCl3 with the heme complexes of the T135V mutant and a Delta221 C-terminal truncated protein yields heme-linked peptides in addition to that from the wild-type reaction. The sequence of the principal labeled peptide from the T135V reaction, 205TAFLLNIQLFEELQELLTHDTK226 , and the lability of the adduct suggest the heme is attached to one of the carboxylic acid residues. A carboxylic acid residue is probably also labeled in the modified peptide 49LVMASLYHIYVALEEEIER67 from the Delta221 truncated protein. Thus, addition of the reductively generated trichloromethyl radical to a heme vinyl group produces a species that alkylates active-site residues. The changes in the alkylated residue caused by the Thr-135 mutation or truncation of the protein places residues in the sequences 49-67 and 205-226 within the active site. Furthermore, this is the first demonstration that heme oxygenase, like cytochrome P450, may catalyze the reductive metabolism of halocarbons and thus contribute to the toxicity of these agents.

Amino Acid Sequence↗

PubMed1998

Reverse-phase capillary high performance liquid chromatography/high performance electrospray ionization mass spectrometry: an essential tool for the characterization of complex glycoprotein digests.

The B-domain of recombinant human Factor VIII comprises 909 amino acids and is extensively N- and O-glycosylated, in that at least 20 different sites are occupied by numerous carbohydrate structures. This domain was incubated with trypsin and subjected to liquid chromatography electrospray ionization mass spectrometry analysis, using an electrospray orthogonal acceleration time-of-flight mass spectrometer as the detector for a capillary reversed phase HPLC separation of the digest. The inherent high mass resolution afforded by this instrument provides both ion charge state determination and high accuracy mass measurement that are of significant advantage in defining such highly complex mixtures.

Amino Acid Sequence↗

PubMed1997

Structural characterization of site-specific N-glycosylation of recombinant human factor VIII by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry.

The present study addresses the site occupancy and the site-specific carbohydrate microheterogeneity of N-linked oligosaccharides in recombinant human factor VIII, expressed in Chinese hamster ovary cells. The four factor VIIIa polypeptides, formed upon incubation with human thrombin, were isolated and separately subjected to proteolysis with trypsin. These tryptic digests were analyzed by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry. Selected ion monitoring of diagnostic carbohydrate ions was utilized to identify glycopeptide-containing chromatographic peaks. Oligomannose and complex carbohydrates were detected at the glycosylation sites of the 50 and the 73 kDa polypeptides, while all the oligosaccharides identified on the B-domain were complex-type structures. Only the 43 kDa polypeptide was found nonglycosylated. These studies established a biantennary core-fucosylated carbohydrate as the major substituent, consistent with the conclusions of the analyses on the entire N-linked carbohydrate pool (Kumar, H. P. M.; Hague, C.; Haley, T.; Starr, C. M.; Besman, M. J.; Lundblad, R.; Baker, D. Biotechnol. Appl. Biochem. 1996, 24, 207-216.). In addition, this mass spectrometric investigation revealed the presence of a complex nonfucosylated oligosaccharide not reported previously for this glycoprotein.

Amino Acid Sequence↗

PubMed1997

Lanthionine-somatostatin analogs: synthesis, characterization, biological activity, and enzymatic stability studies.

A series of cyclic somatostatin analogs containing a lanthionine bridge have been subjected to studies of structure-activity relationships. A direct synthesis of the thioether bridged analog (1) of sandostatin (SMS 201,995) and several lanthionine hexa-, hepta-, and octapeptides was carried out by using the method of cyclization on an oxime resin (PCOR) followed by condensation reactions in solution. The structures of the target peptides were analyzed by liquid secondary ion mass spectrometry (LSIMS) and subjected to high-energy collision-induced dissociation (CID) studies after opening of the peptide ring by proteolytic cleavage. The biological activities of these compounds have been evaluated by assaying their inhibitory potencies for the release of growth hormone (GH) from primary cultures of rat anterior pituitary cells, as well as by their binding affinities to cloned somatostatin receptors (SSTR1-5). The structural modification of sandostatin by introducing a lanthionine bridge resulted in a significantly increased receptor binding selectivity. The lanthionine octapeptide with C-terminal Thr-ol (1) showed similar high affinity for rat SSTR5 compared to somatostatin[1-14] and sandostatin. However, it exhibits about 50 times weaker binding affinity for mSSTR2b than sandostatin. Similarly, the lanthionine octapeptide with the C-terminal Thr-NH2 residue (2) has higher affinity for rSSTR5 than for mSSTR2B. Both peptides (compounds 1 and 2) have much lower potencies for inhibition of growth hormone secretion than sandostatin. This is consistent with their affinities to SSTR2, the receptor which is believed to be linked to the inhibition of growth hormone release by somatostatin and its analogs. The metabolic stability of lanthionine-sandostatin and sandostatin have been studied in rat brain homogenates. Although both compounds have a high stability toward enzymatic degradation, the lanthionine analog has a 2.4 times longer half-life than sandostatin. The main metabolites of both compounds have been isolated and identified by using an in vivo technique (cerebral microdialysis) and mass spectrometry.

Alanine↗

PubMed1997

Synthesis and characterization of histidine-phosphorylated peptides.

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine, and tyrosine have been studied extensively, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins was discovered originally in prokaryotic organisms, but also has been found recently in eukaryotic cells. We describe methods for the synthesis and analysis of phosphohistidine-containing peptides, a prerequisite for the investigation of the role of this posttranslational modification in cellular processes.

Chromatography, High Pressure Liquid↗

PubMed1996

A crosslinked cofactor in lysyl oxidase: redox function for amino acid side chains.

A previously unknown redox cofactor has been identified in the active site of lysyl oxidase from the bovine aorta. Edman sequencing, mass spectrometry, ultraviolet-visible spectra, and resonance Raman studies showed that this cofactor is a quinone. Its structure is derived from the crosslinking of the epsilon-amino group of a peptidyl lysine with the modified side chain of a tyrosyl residue, and it has been designated lysine tyrosylquinone. This quinone appears to be the only example of a mammalian cofactor formed from the crosslinking of two amino acid side chains. This discovery expands the range of known quino-cofactor structures and has implications for the mechanism of their biogenesis.

Amino Acid Sequence↗