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Biomedical subjects

Michael Przybylski

Publications and source records attributed to Michael Przybylski.

At least 19 recordsLinked to original sources
PubMed2007

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome.

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Affinity Labels↗
PubMed2006

N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry.

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.

Animals↗
PubMed2006

A synthetic camel anti-lysozyme peptide antibody (peptibody) with flexible loop structure identified by high-resolution affinity mass spectrometry.

We describe the synthesis and characterisation of the fully functional molecular recognition structure of a 26-amino acid residue peptide antibody, referred to as peptibody, designed from a monoclonal single-domain antibody fragment derived from a camel heavy-chain antibody. The CDR3 region (CDR = complementarity determining region) of the cAbLys3 camel antibody fragment, which binds to the active site of hen eggwhite lysozyme (HEL) and acts as a potent enzyme inhibitor by mimicking an oligosaccharide substrate, was prepared by solid-phase peptide synthesis. To obtain a closed loop-like structure resembling that in the crystal structure, N- and C-terminal cysteine residues were added to the linear peptide and oxidised to a cyclic disulfide-bridged peptide by using dimethylsulfoxide. A further, internal cysteine-12 residue was acetamidomethyl-protected to prevent possible oxidative byproducts. Affinity separation on a lysozyme microcolumn combined with MALDI-TOF mass spectrometry revealed that the peptide resumed high affinity to lysozyme only after deprotection of Cys-12, suggesting the importance of this paratope sequence for epitope recognition. The complex of lysozyme and active peptibody was characterised directly by conducting high-resolution ESI-FTICR mass spectrometry, which provided a molecular comparison of affinities for linear and cyclic peptibodies.

Amino Acid Sequence↗
PubMed2006

Functionalized magnetic micro- and nanoparticles: optimization and application to micro-chip tryptic digestion.

The preparation of an easily replaceable protease microreactor for micro-chip application is described. Magnetic particles coated with poly(N-isopropylacrylamide), polystyrene, poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(glycidyl methacrylate), [(2-amino-ethyl)hydroxymethylen]biphosphonic acid, or alginic acid with immobilized trypsin were utilized for heterogeneous digestion. The properties were optimized, with the constraint of allowing immobilization in a microchannel by a magnetic field gradient. To obtain the highest digestion efficiency, sub-micrometer spheres were organized by an inhomogeneous external magnetic field perpendicularly to the direction of the channel. Kinetic parameters of the enzyme reactor immobilized in micro-chip capillary (micro-chip immobilized magnetic enzyme reactor (IMER)) were determined. The capability of the proteolytic reactor was demonstrated by five model (glyco)proteins ranging in molecular mass from 4.3 to 150 kDa. Digestion efficiency of proteins in various conformations was investigated using SDS-PAGE, HPCE, RP-HPLC, and MS. The compatibility of the micro-chip IMER system with total and limited proteolysis of high-molecular-weight (glyco)proteins was confirmed. It opens the route to automated, high-throughput proteomic micro-chip devices.

Amyloid beta-Peptides↗
PubMed2005

Determination of phosphorus-, copper-, and zinc-containing human brain proteins by LA-ICPMS and MALDI-FTICR-MS.

Human brain proteins containing phosphorus, copper, and zinc were detected directly in protein spots in gels of a human brain sample after separation by two-dimensional gel electrophoresis using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). A powerful laser ablation system with cooled laser ablation chamber was coupled to a double-focusing sector field ICPMS. The separated protein spots in 2D gels were fast screened using the optimized microanalytical LA-ICPMS technique measured at medium mass resolution with a focused laser beam (wavelength, 213 nm; diameter of laser crater, 50 mum; and laser power density, 3 x 10(9) W cm(-2)) with respect to selected three essential elements. Of 176 protein spots in 2D gel from a human brain sample, phosphorus, copper, and zinc were detected in 31, 43, and 49 protein spots, respectively. For the first time, uranium as a naturally occurring radioactive element was found in 20 selected protein spots. The detection limits for P, S, Cu, Zn and U were determined in singular protein spots with 0.0013, 1.29, 0.029, 0.063, and 0.000 01 mg g(-1), respectively. A combination of LA-ICPMS with matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) was applied for the identification of selected protein spots from human brain protein separated by 2D gel electrophoresis. Combining MALDI-FTICR-MS for the structure analysis of metal- and phosphorus-containing human brain proteins with LA-ICPMS, the direct analysis of heteroelements on separated proteins in 2D gels can be performed. For quantification of analytical LA-ICPMS data, the number of sulfur atoms per protein (and following the sulfur concentration) determined by MALDI-FTICR-MS was used for internal standardization. From the known sulfur concentration in protein, the concentration of other heteroelements was calculated. In addition, the number of phosphorylation and the phosphorylation sites of phosphorylated proteins in the human brain sample detected by LA-ICPMS were determined by MALDI-FTICR-MS. This technique allows the study of posttranslational modifications in human brain proteins.

Brain↗
PubMed2005

Implications of the serine protease HtrA1 in amyloid precursor protein processing.

The defining features of the widely conserved HtrA (high temperature requirement) family of serine proteases are the combination of a catalytic protease domain with one or more C-terminal PDZ domains and reversible zymogen activation. Even though HtrAs have previously been implicated in protein quality control and various diseases, including cancer, arthritis, and neuromuscular disorder, the biology of the human family members is not well understood. Our data suggest that HtrA1 is directly involved in the beta-amyloid pathway as it degrades various fragments of amyloid precursor protein while an HtrA1 inhibitor causes accumulation of Abeta in astrocyte cell culture supernatants. Furthermore, HtrA1 colocalizes with beta-amyloid deposits in human brain samples. Potential implications in Alzheimer's disease are discussed.

Aged↗
PubMed2005

Immunoaffinity reactors for prion protein qualitative analysis.

The cellular prion protein (PrPc) represents the substrate for generation of conformational aberrant PrP isoforms which occur in human and animal prion diseases. The published two-dimensional maps of human PrPc show a vast microheterogeneity of this glycoprotein. The main goal of this project was to develop a highly specific immunoaffinity reactor for qualitative analysis of PrP cellular isoforms isolated from brain homogenate, cerebrospinal fluid and other tissues. New techniques for affinity proteomics, carriers and immobilization chemistry were applied. The choice of matrix (chemical and magnetic properties, particle size and distribution, porosity) was the key factor that influenced the quality of the reactor and the nature of final applications. Mouse anti-prion IgGs directed to N-terminal and C-terminal epitopes (residues 23-40 and 147-165) were grafted in different manners to magnetic micro- and nanoparticles particularly developed for micro-CHIP application. High operational and storage stability of affinity reactors with minimized nonspecific absorption were achieved. The quality of the immunoreactors was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and by two-dimensional electrophoresis.

Animals↗
PubMed2005

Statistical evaluation of electrospray tandem mass spectra for optimized peptide fragmentation.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the method of choice for the analysis of complex peptide mixtures. It combines the separation power of nanoflow LC with highly specific sequence analysis, allowing automated peptide sequencing with high resolution and throughput. For peptide fragmentation, the current experimental setup uses predefined parameters based on the mass-to-charge ratio of the individual precursor. Suitable parameters are typically established by empirical evaluation of fragment spectra of individual peptides used as standards. As a result, nonoptimal fragment spectra are obtained if peptides show fragmentation behavior different from these standards, which often result in the loss of sequence-specific fragment ion information. Here we describe a statistical approach for the systematic evaluation of the quality of individual peptide fragment spectra based on the calculation of their arithmetic mean and standard deviation. The method utilizes the dependence of these parameters on the difference in electric potential across the collision cell to determine the value that results in maximum information content. We show that the method is applicable to fragment spectra generated from a variety of multiply-charged tryptic peptides, over a wide concentration range, and on different types of mass analyzers. We also show how this novel approach can be used to define optimized collision energy settings over a wide mass-to-charge range.

Animals↗
PubMed2005

Identification and structural characterisation of carboxy-terminal polypeptides and antibody epitopes of Alzheimer's amyloid precursor protein using high-resolution mass spectrometry.

Alzheimer's disease (AD) is the most common cause for human age-related dementia, characterised by formation of diffuse plaques in brain that are directly involved in AD pathogenesis. The major component of AD plaques is beta-amyloid, a 40 to 42 amino acid polypeptide derived from the amyloid precursor protein (APP) by proteolytic degradation involving the specific proteases, beta-and gamma-secretase acting at the N- and C- terminal cleavage site, respectively. In this study we have prepared polypeptides comprising the carboxy-terminal and transmembrane sequences of APP, by bacterial expression and chemical synthesis, as substrates for studying the C-terminal processing of APP and its interaction with the gamma-secretase complex. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used as a major tool for structure analysis. Immunisation of transgenic mouse models of AD with Abeta42 has been recently shown to be effective to inhibit and disaggregate Abeta-fibrils, and to reduce AD-related neuropathology and memory impairments. However, the mechanism underlying these therapeutic effects has been as yet unclear. Using proteolytic epitope excision from immune complexes in combination with FT-ICR-MS, we identified the epitope recognised by the therapeutically active antibody as the N-terminal Abeta(4-10) sequence; this soluble, nontoxic epitope opens new lead structures for AD vaccine development. A monoclonal antibody (Jonas; JmAb) directed against the cytosolic APP domain was used in studies of APP biochemistry and metabolism. Here we report the identification of the epitope recognised by the JmAb, using the combination of epitope excision and peptide mapping by FT-ICR-MS. The epitope was determined to be located at the C-terminal APP(740-747) sequence; it was confirmed by ELISA binding assays and authentic synthetic peptides and will be an efficient tool in the development of new specific vaccines. These results demonstrate high-resolution FT-ICR-MS as a powerful method for characterising biochemical pathways and molecular recognition structures of APP.

Alzheimer Disease↗
PubMed2005

Structural characterisation of tyrosine-nitrated peptides by ultraviolet and infrared matrix-assisted laser desorption/ionisation Fourier transform ion cyclotron resonance mass spectrometry.

Nitration of tyrosine residues in proteins may occur in cells upon oxidative stress and inflammation processes mediated through generation of reactive nitroxyl from peroxynitrite. Tyrosine nitration from oxidative pathways may generate cytotoxic species that cause protein dysfunction and pathogenesis. A number of protein nitrations in vivo have been reported and some specific Tyrosine nitration sites have been recently identified using mass spectrometric methods. High-resolution Fourier transform ion cyclotron resonance mass spectrometry (MALDI) FT-ICR-MS) is shown here to be a highly efficient method in the determination of protein nitrations. Following the identification of nitration of the catalytic site Tyr-430 residue of bovine prostacyclin synthase, we synthesised several model peptides containing both unmodified tyrosine and 3-nitro-tyrosine residues, using solid-phase peptide synthesis (SPPS). The structures of the nitrotyrosine peptides were characterised both by ESI- and by matrix-assisted laser desorption/ionisation (MALDI)-FT-ICR-MS, using a standard ultraviolet (UV) nitrogen nitrogen laser and a 2.97 microm Nd-YAG infrared laser. Using UV-MALDI-MS, 3-nitrotyrosyl-peptides were found to undergo extensive photochemical fragmentation at the nitrophenyl group, which may hamper or prevent the unequivocal identification of Tyr-nitrations in cellular proteins. In contrast, infrared-MALDI-FT-ICR-MS did not produce fragmentation of molecular ions of Tyr-nitrated peptides.

Amino Acid Sequence↗
PubMed2004

Immunoproteasomes down-regulate presentation of a subdominant T cell epitope from lymphocytic choriomeningitis virus.

The cytotoxic T cell response to pathogens is usually directed against a few immunodominant epitopes, while other potential epitopes are either subdominant or not used at all. In C57BL/6 mice, the acute cytotoxic T cell response against lymphocytic choriomeningitis virus is directed against immunodominant epitopes derived from the glycoprotein (gp33-41) and the nucleoprotein (NP396-404), while the gp276-286 epitope remains subdominant. Despite extensive investigations, the reason for this hierarchy between epitopes is not clear. In this study, we show that the treatment of cells with IFN-gamma enhanced the presentation of gp33-41, whereas presentation of the gp276-286 epitope from the same glycoprotein was markedly reduced. Because proteasomes are crucially involved in epitope generation and because IFN-gamma treatment in vitro and lymphocytic choriomeningitis virus infection in vivo lead to a gradual replacement of constitutive proteasomes by immunoproteasomes, we investigated the role of proteasome composition on epitope hierarchy. Overexpression of the active site subunits of immunoproteasomes LMP2, LMP7, and MECL-1 as well as overexpression of LMP2 alone suppressed the presentation of the gp276-286 epitope. The ability to generate gp276-286-specific CTLs was enhanced in LMP2- and LMP7-deficient mice, and macrophages from these mice showed an elevated presentation of this epitope. In vitro digests demonstrated that fragmentation by immunoproteasomes, but not constitutive proteasomes led to a preferential destruction of the gp276 epitope. Taken together, we show that LMP2 and LMP7 can at least in part determine subdominance and shape the epitope hierarchy of CTL responses in vivo.

Amino Acid Sequence↗
PubMed2004

Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments.

The newly developed immobilized enzyme reactors (IMERs) with proteolytic enzymes chymotrypsin, trypsin or papain were used for specific fragmentation of high molecular-mass and heterogeneous glycoproteins immunoglobulin G (IgG) and crystallizable fragment of IgG (Fc). The efficiency of splitting or digestion were controlled by RP-HPLC. The specificity of digestion by trypsin reactor was controlled by MS. IMERs (trypsin immobilized on magnetic microparticles focused in a channel of magnetically active microfluidic device) was used for digestion of the whole IgG molecule. The sufficient conditions for IgG digestion in microfluidic device (flow rate, ratio S:E, pH, temperature) were optimized. It was confirmed that the combination of IMERs with microfluidic device enables efficient digestion of highly heterogeneous glycoproteins such as IgG in extremely short time and minimal reaction volume.

Amino Acid Sequence↗
PubMed2004

Zinc binding agonist effect on the recognition of the beta-amyloid (4-10) epitope by anti-beta-amyloid antibodies.

Amyloid plaques associated to Alzheimer's disease present a high content of zinc ions. We previously showed that the N-terminal region of the amyloid peptide Abeta constitutes an autonomous zinc-binding domain. This region encompasses the previously identified epitope Abeta(4-10) targeted by antibodies capable to reduce amyloid deposition, but the influence of Abeta/Zn binding on the epitope recognition remains unknown. We demonstrate here the effect of Zn2+ ions on the recognition of peptides sharing the sequence of the Abeta N-terminal domain, by two monoclonal antibodies recognizing the beta-amyloid(4-10) epitope. The presence of Zn2+, but not of other cations, increased the recognition of the (1-16) peptide, while it was without effect on the recognition of the (1-10) peptide. These findings show a zinc-induced conformational change of the (1-16)-N-terminal region of AP3, which results in a better accessibility of the Abeta(4-10) epitope to the anti-Abeta antibodies, and suggest a role of zinc in epitope-based vaccination approaches.

Amyloid beta-Peptides↗
PubMed2004

Polypeptide conjugates comprising a beta-amyloid plaque-specific epitope as new vaccine structures against Alzheimer's disease.

Immunotherapeutic approaches designed to induce a humoral immune response have recently been developed for possible vaccination to the treatment of Alzheimer's disease (AD). Based on the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, branched polypeptide conjugates with this epitope peptide were synthesized and characterized. In order to produce immunogenic constructs, the Abeta(4-10) epitope alone or together with a promiscuous T-helper cell epitope peptide (FFLLTRILTIPQSLD) were attached via thioether linkage to different branched chain polymeric polypeptides with Ser or Glu in the side chains. A single peptide containing both an Abeta(4-10) and T-helper cell epitope, joined by a dipeptide Cys-Acp spacer, was also attached through the thiol function to chloroacetylated poly[Lys(Seri-DL-Alax)] (SAK). Comparative binding studies of the conjugates with a monoclonal antibody against the beta-amyloid(1-17) peptide in mice were performed by direct ELISA. The conformational preferences of carriers and conjugates in water and in a 9:1 trifluoroethanol:water mixture (v/v) was analyzed by CD spectroscopy. Experimental data showed that the chemical nature of the carrier macromolecule, and the attachment site of the epitope to the carrier, have significant effects on antibody recognition, but have no marked influence on the solution conformation of the conjugates.

Alzheimer Disease↗
PubMed2004

High resolution mass spectrometric alveolar proteomics: identification of surfactant protein SP-A and SP-D modifications in proteinosis and cystic fibrosis patients.

In the present study, one- and two-dimensional gel electrophoresis combined with high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) have been applied as powerful approaches for the proteome analysis of surfactant proteins SP-A and SP-D, including identification of structurally modified and truncation forms, in bronchoalveolar lavage fluid from patients with cystic fibrosis, chronic bronchitis and pulmonary alveolar proteinosis. Highly sensitive micropreparation techniques were developed for matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS analysis which provided the identification of surfactant proteins at very low levels. Owing to the high resolution, FT-ICR MS was found to provide substantial advantages for the structural identification of surfactant proteins from complex biological matrices with high mass determination accuracy. Several protein bands corresponding to SP-A and SP-D were identified by MALDI-FT-ICR MS after electrophoretic separation by one- and two-dimensional gel electrophoresis, and provided the identification of structural modifications (hydroxy-proline) and degradation products. The high resolution mass spectrometric proteome analysis should facilitate the unequivocal identification of subunits, aggregations, modifications and degradation products of surfactant proteins and hence contribute to the understanding of the mechanistic basis of lung disease pathogenesis.

Bronchoalveolar Lavage Fluid↗
PubMed2004

Synthesis and structural characterization of bioactive peptide conjugates using thioether linkage approaches.

Applications of cysteine-insertion and thioether linkage approaches to the preparation of a number of bioactive peptide conjugates are reported. Peptides containing epitopes from (i) herpes simplex virus type 1 glycoprotein D, (ii) a specific N-terminal beta-amyloid epitope recognized by therapeutically active antibodies, and (iii) a GnRH-III peptide from sea lamprey with antitumour activity, were elongated with Cys residues and attached to a chloroacetylated tetratuftsin derivative carrier via a thioether linkage either directly, or by insertion of a spacer. The structures and molecular homogeneity of all the peptide conjugates were ascertained by HPLC, MALDI and electrospray mass spectrometry. The use of a spacer such as an oligoglycine or GFLG-tetrapeptide gave an increased yield in the conjugation reaction and enhanced reaction rates. In the formation of cysteinyl-thioether linkages, it was found that the position of flanking Cys residues markedly influenced the conjugation reaction and the formation of intermolecular epitope disulfide-dimers. C-terminal Cys residues gave thioether conjugates with significantly diminished epitope-dimerization, while Cys at the N-terminal caused rapid disulfide-dimerization, thereby preventing efficient conjugation.

Acetates↗
PubMed2004

Phosphorylation by protein kinase CK2 changes the DNA binding properties of the human chromatin protein DEK.

We have examined the posttranslational modification of the human chromatin protein DEK and found that DEK is phosphorylated by the protein kinase CK2 in vitro and in vivo. Phosphorylation sites were mapped by quadrupole ion trap mass spectrometry and found to be clustered in the C-terminal region of the DEK protein. Phosphorylation fluctuates during the cell cycle with a moderate peak during G(1) phase. Filter binding assays, as well as Southwestern analysis, demonstrate that phosphorylation weakens the binding of DEK to DNA. In vivo, however, phosphorylated DEK remains on chromatin. We present evidence that phosphorylated DEK is tethered to chromatin throughout the cell cycle by the un- or underphosphorylated form of DEK.

Amino Acid Sequence↗