Michael Jürgens - Academia.edu (original) (raw)

Papers by Michael Jürgens

Journal of Mass Spectrometry, Feb 1, 2005

Recent work on protein and peptide biomarker patterns revealed the difficulties in identifying th... more Recent work on protein and peptide biomarker patterns revealed the difficulties in identifying their molecular components, which is indispensable for validation of the biological context. Cerebrospinal fluid and brain tissue are used as sources to discover new biomarkers, e.g. for neurodegenerative diseases. Many of these biomarker candidates are peptides with a molecular mass of <10 kDa. Their identification is favourably achieved with a 'top-down' approach, because this requires less purification and an enzymatic cleavage will often not yield enough specific fragments for successful database searches. Here, we describe an approach using quadrupole time-of-flight mass spectrometry (TOFMS) as a highly efficient mass spectrometric purification and identification tool after off-line decomplexation of biological samples by liquid chromatography. After initial peptidomic screening with matrix-assisted laser desorption/ionization (MALDI) TOFMS, the elution behaviour in chromatography and the exact molecular mass were used to locate the same signals in nanoelectrospray measurements. Most of the peaks detected in MALDI-TOFMS could be retrieved in nanoelectrospray quadrupole TOFMS. Suitable collision energies for informative fragment spectra were investigated for different parent ions, charge states and molecular masses. After collision-induced dissociation, the resulting fragmentation data of multiply charged ions can become much more complicated than those derived from tryptic peptide digests. However, the mass accuracy and resolution of quadrupole TOF instruments results in high-quality data suitable for determining peptide sequences. The protein precursor, proteolytic processing and post-translational modifications were identified by automated database searches. This is demonstrated by the exemplary identifications of thymosin beta-4 (5.0 kDa) and NPY (4.3 kDa) from rat hypothalamic tissue and ubiquitin (8.6 kDa) from human cerebrospinal fluid. The high data quality should also allow for de novo identification. This methodology is generally applicable for peptides up to a molecular mass of about 10 kDa from body fluids, tissues or other biological sources.

Rapid Communications in Mass Spectrometry, Aug 3, 2001

Combinatorial Chemistry & High Throughput Screening, Dec 1, 2005

Macromolecular Theory and Simulations, 2002

Aspects of applying n-pulse periodic initiation in pulsed laser polymerization/size-exclusion chr... more Aspects of applying n-pulse periodic initiation in pulsed laser polymerization/size-exclusion chromatography (PLP/SEC) experiments are studied via simulation of molecular weight distributions (MWDs). In n-pulse periodic PLP/SEC, sequences of n laser pulses at successive time intervals Δt 1 up to Δt n are periodically applied. With the dark time intervals being suitably chosen, n-modal MWDs with n well separated peaks occur. The n-pulse periodic PLP/SEC method has the potential for providing accurate propagation rate coefficients, k p . Among several measures for k p , the differences in molecular weights at the MWD peak positions vield the best estimate of k p under conditions of medium and high pulse laser-induced free-radical concentration. Deducing k p from n dark time intervals (corresponding to n regions of free-radical chain length) within one experiment at otherwise identical PLP/SEC conditions allows addressing in more detail a potential chain-length dependence of k p . Simulations are compared with experimental data for 2-pulse periodic polymerization of methyl methacrylate.

Macromolecular Symposia, 2004

Free‐radical terpolymerizations of styrene, methyl methacrylate, and glycidyl methacrylate were c... more Free‐radical terpolymerizations of styrene, methyl methacrylate, and glycidyl methacrylate were carried out in a tubular reactor in the presence of 20 wt.% CO2 at temperatures between 120 and 180°C and pressures of 300 and 350 bar. The number average molecular weights, MN, were mostly between 2000 and 3000 g·mol−1 and polydispersity indices around 2. In part of the experiments molecular weights were controlled by n‐dodecyl mercaptan serving as the chain‐transfer agent. PREDICI modeling indicates that the targeted molecular weights of MN∼2500 g·mol−1 and polydispersities around 2 may also be reached by using an initiator cocktail, a mixture of two initiators with significantly different decomposition rate coefficients. The predictions are confirmed experimentally.

Macromolecular Chemistry and Physics, 2004

The Journal of Supercritical Fluids, 2006

Terpolymerizations of styrene, methyl methacrylate, and glycidyl methacrylate in solution with su... more Terpolymerizations of styrene, methyl methacrylate, and glycidyl methacrylate in solution with supercritical CO 2 were carried out in a tubular reactor up to almost complete monomer conversion. The residence time behavior of the setup was investigated by injecting a tracer pulse and measuring the tracer concentration at the reactor exit via in-line FT-near infrared (NIR) spectroscopy. Molecular weight control during polymerization was achieved either by initiator concentration or by the addition of a catalytic chain-transfer agent. Number average molecular weights were adjusted to about 2500 g mol −1 to yield products which are suitable components for powder coating applications.

Industrial & Engineering Chemistry Research, 2003

Styrene−methyl methacrylate−glycidyl methacrylate terpolymers to be used as binders in powder coa... more Styrene−methyl methacrylate−glycidyl methacrylate terpolymers to be used as binders in powder coating materials were synthesized in a homogeneous fluid phase containing 20 wt % CO2. Reactions carried out at 120 °C and 350 bar yield almost complete monomer conversion. The molecular weights were around 3000 g·mol-1 with a polydispersity index of 1.7. Molecular weight control was achieved by n-dodecyl mercaptan or by a catalytic chain-transfer agent. Simulations of the cumulative and differential copolymer composition are in excellent agreement with experimentally derived values.

Chemie Ingenieur Technik, 2001

Phase behavior measurements for terpolymers of styrene, methyl methacrylate (MMA), and glycidyl m... more Phase behavior measurements for terpolymers of styrene, methyl methacrylate (MMA), and glycidyl methacrylate (GMA) in a mixture of supercritical (sc) CO 2 and acetone show that, in case of low molecular weight material (MW~5000 g´mol ±1), significant amounts of the polymer may be dissolved at pressures around 300 bar. Based on this finding, terpolymerizations of styrene, MMA, and functional methacrylates such as GMA and 2-hydroxypropyl methacrylate have been carried out in scCO 2. Almost complete monomer conversion may be reached, e.g. at 120 C, 350 bar and in the presence of 20 wt.% scCO 2. The reaction kinetics and the polymer molecular weight have been modeled using the program PREDICI Ò. The modeling is exclusively based on kinetic coefficients from separate pulsed laser experiments.

Journal of Chromatography B, Dec 1, 2002

Peptides, such as many hormones, cytokines and growth factors play a central role in biological p... more Peptides, such as many hormones, cytokines and growth factors play a central role in biological processes. Furthermore, as degradation products and processed forms of larger proteins they are part of the protein turnover. Thus, they can reflect disease-related changes in an organism's homeostasis in several ways. Since two-dimensional gel electrophoresis is restricted to analysis and display of proteins with relative molecular masses above 5000, we developed Differential Peptide Display (DPD), a new technology for analysis and visualization of peptides. Here we describe its application to cerebrospinal fluid of three subjects without a disease of the central nervous system (CNS) undergoing routine myelography and of two patients suffering from a primary CNS lymphoma. Peptides with a relative molecular mass below 20 000 were extracted and analysed by a combination of chromatography and mass spectrometry. The peptide pattern of a sample was depicted as a multi-dimensional peptide mass fingerprint with each peptide's position being characterized by its molecular mass and chromatographic behaviour. Such a fingerprint of a CNS sample consists of more than 6000 different signals. Data analysis of peptide patterns from patients with CNS lymphoma compared to controls revealed obvious differences regarding the peptide content of the samples. By analysing peptides within a mass range of 750-20 000, DPD extends 2D gel electrophoresis, thus offering the chance to investigate CNS diseases on the level of peptides. This represents a new approach for diagnosis and possible therapy.

$Research paper thumbnail of Composition of the peptide fraction in human blood plasma: database of circulating human peptides$

Journal of Chromatography B: Biomedical Sciences and Applications, 1999

A database was established from human hemofiltrate (HF) that consisted of a mass database and a s... more A database was established from human hemofiltrate (HF) that consisted of a mass database and a sequence database, with the aim of analyzing the composition of the peptide fraction in human blood. To establish a mass database, all 480 fractions of a peptide bank generated from HF were analyzed by MALDI-TOF mass spectrometry. Using this method, over 20 000 molecular masses representing native, circulating peptides were detected. Estimation of repeatedly detected masses suggests that approximately 5000 different peptides were recorded. More than 95% of the detected masses are smaller than 15 000, indicating that HF predominantly contains peptides. The sequence database contains over 340 entries from 75 different protein and peptide precursors. 55% of the entries are fragments from plasma proteins (fibrinogen A 13%, albumin 10%, b2-microglobulin 8.5%, cystatin C 7%, and fibrinogen B 6%). Seven percent of the entries represent peptide hormones, growth factors and cytokines. Thirty-three percent belong to protein families such as complement factors, enzymes, enzyme inhibitors and transport proteins. Five percent represent novel peptides of which some show homology to known peptide and protein families. The coexistence of processed peptide fragments, biologically active peptides and peptide precursors suggests that HF reflects the peptide composition of plasma. Interestingly, protein modules such as EGF domains (meprin Aa-fragments), somatomedin-B domains (vitronectin fragments), thyroglobulin domains (insulin like growth factor-binding proteins), and Kazal-type inhibitor domains were identified. Alignment of sequenced fragments to their precursor proteins and the analysis of their cleavage sites revealed that there are different processing pathways of plasma proteins in vivo.

The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass... more The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass spectrometry. The N-terminal disintegrin-like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues~XIII and XVI! that form an additional disulfide bond that is characteristically found in the disintegrin-like domains of cellular metalloproteinases~ADAMs! and PIII snake venom Zn-metalloproteinases~SVMPs!. The C-terminal cysteine-rich domain of catrocollastatin-C contains five disulfide bonds between nearest-neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin-like and cysteine-rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed.

Biochemical Journal, 2000

The disulphide-bond pattern of the heterodimeric disintegrin EMF-10, a potent and selective integ... more The disulphide-bond pattern of the heterodimeric disintegrin EMF-10, a potent and selective integrin α & β " antagonist from Eristocophis macmahoni venom, was established by combination of amino-acid analysis, N-terminal sequencing and collisioninduced dissociation by nanoelectrospray ionization quadrupole ion-trap MS of fragments isolated by reversed-phase HPLC after degradation of EMF-10 with oxalic acid. Each EMF-10 subunit contains four intrachain disulphide bonds. Two interchain cystine residues join the EMF-10 polypeptides. The intrachain linkages are conserved in monomeric disintegrins. A molecular model of EMF-10 was built using averaged NMR coordinates of flavo-Abbreviations used : TFA, trifluoroacetic acid ; MALDI-TOF, matrix-assisted laser-desorption ionization-time-of-flight ; QIT, quadrupole ion-trap ; CID, collision-induced dissociation ; nanoES, nanoelectrospray. 1 To whom correspondence should be addressed (e-mail jcalvete!ibv.csic.es). ridin as a template. The active hairpin loops of the EMF-10 subunits occupy opposite locations at the ends of an elongated disulphide-bond ladder. In the EMF-10 model the N-terminal polypeptide of EMF-10B is close to the RGD-loop of the EMF-10A subunit, suggesting that the N-terminal region of the Bsubunit could potentially influence the biological activity of the A-subunit.

Journal of Chromatography A, 1997

Human hemofiltrate (HF) is a source for the purification of circulating regulatory peptides. HF i... more Human hemofiltrate (HF) is a source for the purification of circulating regulatory peptides. HF is obtained in large quantities during treatment of patients suffering from chronic renal failure. We have developed a large-scale method for separating peptides from amounts up to 10,000 1 HF into 300 fractions in a standardized two-step procedure, employing cation-exchange separation, followed by reversed-phase chromatography. These fractions represent a peptide bank containing bioactive, desalted and lyophilized peptides of blood. Screening for and isolation of regulatory human peptides is simplified by using this peptide bank.

Journal of Number Theory, 2017