Biomedical subjects

Peter Juhasz

Publications and source records attributed to Peter Juhasz.

9 recordsLinked to original sources

PubMed2005

Quantitative proteomic analysis of fibroblast nuclear proteins after stimulation with mitogen activated protein kinase inhibiting heparan sulfate.

Certain structures of heparan sulfate (HS) inhibit cell proliferation of fibroblasts. Whether this inhibition is dependent on inhibition of mitogenic signaling pathways or nuclear translocation of HS is unknown. In this study we investigated possible mechanism(s) and structural requirements by which antiproliferative glycosaminoglycans exert their effects on mitogen-activated protein kinase (MAP kinase) phosphorylation, a key intermediate in cell signaling, followed by quantitative proteomic analysis of nuclear proteins by stable isotope coded affinity tags, multidimensional chromatography and tandem mass spectrometry. Serum starved human lung fibroblasts were stimulated with serum, platelet derived growth factor (PDGF-BB) or epidermal growth factor (EGF) in the presence of structurally different glycosaminoglycans. Antiproliferative heparan sulfate with a high content of 2-O-sulfated iduronic acid (IdoA-2SO4) and heavily sulfated glucosamine, and the structurally related glycosaminoglycan heparin inhibited significantly serum stimulated MAP kinase phosphorylation, by at least 80% when stimulated by serum and HS6. We hypothesized that the inhibition of the MAP kinase pathway will have effect in the nuclear proteome. Therefore an isotope coded affinity tag (ICAT) reagent labeling of nuclear proteins and tandem mass spectrometry was applied, resulting in the identification and quantification of 206 proteins. Several nuclear proteins were found to be induced or repressed due to HS stimulation, where the repression EBNA-2 co-activator and the induction of PML protein were of special interest. These results show that heparan sulfate with high content of (IdoA-2SO4) and heavily sulfated glucosamine specifically inhibits MAP kinase activation with a subsequent change in the nuclear proteome of the fibroblast.

Binding Sites↗

PubMed2005

Identification of Amadori-modified plasma proteins in type 2 diabetes and the effect of short-term intensive insulin treatment.

OBJECTIVE: Growing evidence supports that nonenzymatic glycation products may cause hyperglycemia-induced diabetes complications. Amadori-modified proteins are the intermediate products of nonenzymatic glycation and constitute the forms of glycated proteins in diabetes. The objective of the current study was to utilize two-dimensional gel electrophoresis, Western blot, and mass spectrometry to identify Amadori-modified plasma proteins in type 2 diabetic patients with poor glycemic control and assess the impact of short-term insulin treatment on the glycation of these proteins. RESEARCH DESIGN AND METHODS: We compared eight type 2 diabetic subjects (aged 56 +/- 3 years and BMI 29.7 +/- 0.9 kg/m(2)) with an average diabetes duration of 8.5 years (range 3-19) with equal numbers of weight-matched (aged 56 +/- 2 years and BMI 30.1 +/- 10.0 kg/m(2)) and lean (aged 58 +/- 2 years and BMI 25 +/- 00.5 kg/m(2)) nondiabetic subjects who have no first-degree relatives with diabetes. Two separate blood samples were collected from the type 2 diabetic subjects, one following 2 weeks of withdrawal of all antidiabetic medications (T(2)D-; plasma glucose 12.6 +/- 1.0 mmol/l) and another following 10 days of intensive insulin treatment (T(2)D+; plasma glucose 5.5 +/- 0.2 mmol/l). Plasma proteins were separated using single and two-dimensional gel electrophoresis. Western blot analysis was performed, and several proteins, which reacted with the Amadori-antibody (1-deoxyfructosyl lysine), were identified by tandem mass spectrometry. RESULTS: No significant differences in the glycation of proteins between the obese and lean groups were noted, but type 2 diabetic patients had several proteins with higher glycation than the control groups. We identified 12 plasma proteins with reduced reaction to the anti-Amadori antibody upon intensive insulin treatment. A significant (P < 0.03) difference in Amadori modification was observed between the T(2)D- and control subjects for all these proteins except the Ig light chain. Insulin treatment reduced Amadori modification of albumin (23.2%, P < 0.02), fibrin (34.6%, P < 0.001), Ig heavy chain constant region (20.7%, P < 0.05), transferrin (25.4%, P < 0.04), and Ig light chain (13%, P < 0.02). In addition, Western blot analysis of two-dimensional gel electrophoresis identified alpha-fibrinogen precursor, beta-fibrinogen precursor, fibrinogen gamma-B chain precursor, hemopexin, vitamin D binding protein, and serine protease inhibitor as proteins with a reduced reaction to anti-Amadori antibody upon intensive insulin treatment. CONCLUSIONS: The current approach offers the opportunity to identify Amadori modification of many proteins that may cause functional alterations and offers the potential for monitoring short-term glycemic control in diabetic patients.

Blood Glucose↗

PubMed2004

Effect of solvent composition on signal intensity in liquid chromatography-matrix-assisted laser desorption ionization experiments.

The use of reversed phase liquid chromatography for the preparation of complex peptide mixtures for analysis by matrix assisted laser desorption ionization mass spectrometry has led to the observation of the critical importance of the matrix/analyte formulation in regards to the percent organic solvent in the mixture. This paper outlines the study using liquid chromatography to systematically vary the acetonitrile concentration in the formulation used for MALDI spot preparation to examine the impact the parameter has on analyte signal intensity. The results show that for five of six peptides tested across a wide mass range a formulation of approximately 75% acetonitrile is optimal for average MALDI signal intensity as determined on both time-of-flight and quadropole mass spectrometers. Examination of the individual spots shows that the organic solvent content in formulation significantly affects parameters such as crystal density and morphology.

Chromatography, Liquid↗

PubMed2004

Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.

We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Delta and xrn1Delta mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5' to 3' decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.

Cations↗

PubMed2004

Depth of proteome issues: a yeast isotope-coded affinity tag reagent study.

As a test case for optimizing how to perform proteomics experiments, we chose a yeast model system in which the UPF1 gene, a protein involved in nonsense-mediated mRNA decay, was knocked out by homologous recombination. The results from five complete isotope-coded affinity tag (ICAT) experiments were combined, two using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) and three using electrospray MS/MS. We sought to assess the reproducibility of peptide identification and to develop an informatics structure that characterizes the identification process as well as possible, especially with regard to tenuous identifications. The cleavable form of the ICAT reagent system was used for quantification. Most proteins did not change significantly in expression as a consequence of the upf1 knockout. As expected, the Upf1 protein itself was down-regulated, and there were reproducible increases in expression of proteins involved in arginine biosynthesis. Initially, it seemed that about 10% of the proteins had changed in expression level, but after more thorough examination of the data it turned out that most of these apparent changes could be explained by artifacts of quantification caused by overlapping heavy/light pairs. About 700 proteins altogether were identified with high confidence and quantified. Many peptides with chemical modifications were identified, as well as peptides with noncanonical tryptic termini. Nearly all of these modified peptides corresponded to the most abundant yeast proteins, and some would otherwise have been attributed to "single hit" proteins at low confidence. To improve our confidence in the identifications, in MALDI experiments, the parent masses for the peptides were calibrated against nearby components. In addition, five novel parameters reflecting different aspects of identification were collected for each spectrum in addition to the Mascot score that was originally used. The interrelationship between these scoring parameters and confidence in protein identification is discussed.

Amino Acid Sequence↗

PubMed2003

Nanocapillary liquid chromatography interfaced to tandem matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry: mapping the nuclear proteome of human fibroblasts.

Miniaturized liquid chromatography nanoseparation in combination with minigel fractionation of human primary cell nuclei is presented. We obtained high-sensitivity and high-throughput identification of expressed proteins by subcellular fractionation and nanocapillary liquid chromatography interfaced to both electrospray ionization (ESI)- and matrix-assisted laser desorption/ionisation (MALDI) tandem mass spectrometry. The reversed-phase nanocapillary eluents were applied directly onto the MALDI target plate as discrete crystal spots using in-line matrix infusion. When working with primary cells, only a limited amount of sample is available. To maximize the number of identified proteins from a restricted amount of sample, miniaturized sample preparation protocols and nanoflow separation is a necessity, especially when working with low-abundant proteins. From the same isolated nuclear sample, complementary separation of intact proteins by two-dimensional (2-D) gel electrophoresis was made. In total 594 gene products from the nuclear preparations were identified out of which 261 were unique. Several proteins involved in transcriptional events were identified such as TATA-binding protein, EBNA-co-activator, and interleukin enhancer binding proteins, indicating that sufficient proteomic depth is obtained to study transcriptional controlling events. Our results suggest that by sample prefractionation and downscaled nanoflow separation along with a combined mass spectrometry strategy, it is possible to identify a large number of nuclear proteins from human primary cells. These findings are of particular importance due to the disease link of these targets cells.

Cell Nucleus↗

PubMed2002

The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry.

Mass spectrometry has become the technology of choice for detailed identification of proteins in complex mixtures. Although electrophoretic separation, proteolytic digestion, mass spectrometric analysis of unseparated digests, and database searching have become standard methods in widespread use, peptide sequence information obtained by collision-induced dissociation and tandem mass spectrometry is required to establish the most comprehensive and reliable results. Most tandem mass spectrometers in current use employ electrospray ionization. In this work a novel tandem mass spectrometer employing matrix-assisted laser desorption ionization-time-of-flight/time-of-flight operating at 200 Hz has been used to identify proteins interacting with known nucleoporins in the nuclear pore complex of Saccharomyces cerevisiae. Proteins interacting with recombinant proteins as bait were purified from yeast extracts and then separated by one-dimensional SDS-PAGE. Although peptide mass fingerprinting is sometimes sufficient to identify proteins, this study shows the importance of employing tandem mass spectrometry for identifying proteins in mixtures or as covalently modified forms. The rules for incorporating these features into MS-Tag are presented. In addition to providing an evaluation of the sensitivity and overall quality of collision-induced dissociation spectra obtained, standard conditions for ionization and fragmentation have been selected that would allow automatic data collection and analysis, without the need to adjust parameters in a sample-specific fashion. Other considerations essential for successful high throughput protein analysis are discussed.

Amino Acid Sequence↗

PubMedDate not supplied

Off-line coupling of high-resolution capillary electrophoresis to MALDI-TOF and TOF/TOF MS.

High-resolution capillary electrophoresis has been coupled to MALDI-TOF and TOF/TOF MS through off-line vacuum deposition onto standard stainless steel MALDI targets. This off-line approach allowed the decoupling of the separation from the MS analysis, thus allowing each to be independently optimized in terms of time. Using BSA tryptic digest as a model sample, the deposited streaks, roughly 100-microm wide, were first analyzed in the MS mode, consuming only a fraction of the sample. After data analysis, segments of the deposited trace, containing unidentified peptides, as well as several species chosen for sequence confirmation, were reanalyzed in the MS/MS mode using MALDI-TOF/TOF MS. Additionally, it is shown that the shot-to-shot reproducibility of the vacuum-deposited trace (5% RSD) is 1 order of magnitude lower than that found for the standard dried droplet method. Moreover, a linear dependence of signal intensities (relative to an internal standard) over 3 orders of magnitude was found for a peptide sample with concentrations ranging from 1 to 1000 nM. This paper demonstrates the potential of off-line coupling of high-resolution separations to MALDI-MS and MALDI-MS/MS using vacuum deposition for the analysis of complex peptide mixtures from protein digests.

Electrophoresis, Capillary↗

PubMedDate not supplied

Comparative study of [Three] LC-MALDI workflows for the analysis of complex proteomic samples.

Large-scale proteomic analyses frequently rely on high-resolution peptide separation of digested protein mixtures in multiple dimensions to achieve accuracy in sample detection and sensitivity in dynamic range of coverage. This study was undertaken to demonstrate the feasibility of MALDI MS/MS with off-line coupling to HPLC for the analysis of whole cell lysates of wild-type yeast by three different workflows: SCX-RPHPLC-MS/MS, high-pH SAX-RPHPLC-MS/MS and RP (protein)-SCX-RPHPLC-MS/MS. The purpose of these experiments was to demonstrate the effect of a workflow on the end results in terms of the number of proteins detected, the average peptide coverage of proteins, and the number of redundant peptide sequencing attempts. Using 60 microg of yeast lysate, minor differences were seen in the number of proteins detected by each method (800-1200). The most significant differences were observed in redundancy of MS/MS acquisitions.

Cations↗