Publications by authors named "Cornelia Koy"

27 Publications

  • Page 1 of 1

Profiling of intact blood proteins by matrix-assisted laser desorption/ionization mass spectrometry without the need for freezing - Dried serum spots as future clinical tools for patient screening.

Rapid Commun Mass Spectrom 2021 Jul;35(14):e9121

Proteome Center Rostock, Medical Faculty and Natural Science Faculty, University of Rostock, Schillingallee 69, Rostock, 18057, Germany.

Rationale: To open up new ways for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based patient screening, blood serum is the most preferred specimen because of its richness in patho-physiological information and due to ease of collection. To overcome deleterious freeze/thaw cycles and to reduce high costs for shipping and storage, we sought to develop a procedure which enables MALDI-MS protein profiling of blood serum proteins without the need for serum freezing.

Methods: Blood sera from patients/donors were divided into portions which after pre-incubation were fast frozen. Thawed aliquots were deposited on filter paper discs and air-dried at room temperature. Intact serum proteins were eluted with acid-labile detergent-containing solutions and were desalted by employing a reversed-phase bead system. Purified protein solutions were screened by MALDI-MS using standardized instrument settings.

Results: MALDI mass spectra from protein solutions which were eluted from filter paper discs and desalted showed on average 25 strong ion signals (mass range m/z 6000 to 10,000) from intact serum proteins (apolipoproteins, complement proteins, transthyretin and hemoglobin) and from proteolytic processing products. Semi-quantitative analysis of three ion pairs: m/z 6433 and 6631, m/z 8205 and 8916, as well as m/z 9275 and 9422, indicated that the mass spectra from either pre-incubated fast-frozen serum or pre-incubated dried serum spot eluted serum contained the same information on protein composition.

Conclusions: A workflow that avoids the conventional cold-chain and yet enables the investigation of intact serum proteins and/or serum proteolysis products by MALDI-MS profiling was developed. The presented protocol tremendously broadens the clinical application of MALDI-MS and simultaneously allows a reduction in the costs for storage and shipping of serum samples. This will pave the way for clinical screening of patients also in areas with limited access to health care systems, and/or specialized laboratories.
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http://dx.doi.org/10.1002/rcm.9121DOI Listing
July 2021

Mass Spectrometric Analysis of Antibody-Epitope Peptide Complex Dissociation: Theoretical Concept and Practical Procedure of Binding Strength Characterization.

Molecules 2020 Oct 17;25(20). Epub 2020 Oct 17.

Proteome Center Rostock, University Medicine Rostock, 18059 Rostock, Germany.

Electrospray mass spectrometry is applied to determine apparent binding energies and quasi equilibrium dissociation constants of immune complex dissociation reactions in the gas phase. Myoglobin, a natural protein-ligand complex, has been used to develop the procedure which starts from determining mean charge states and normalized and averaged ion intensities. The apparent dissociation constant KD m0g#= 3.60 × 10 for the gas phase heme dissociation process was calculated from the mass spectrometry data and by subsequent extrapolation to room temperature to mimic collision conditions for neutral and resting myoglobin. Similarly, for RNAse S dissociation at room temperature a KD m0g#= 4.03 × 10 was determined. The protocol was tested with two immune complexes consisting of epitope peptides and monoclonal antibodies. For the epitope peptide dissociation reaction of the FLAG peptide from the antiFLAG antibody complex an apparent gas phase dissociation constant KD m0g#= 4.04 × 10 was calculated. Likewise, an apparent KD m0g#= 4.58 × 10 was calculated for the troponin I epitope peptide-antiTroponin I antibody immune complex dissociation. Electrospray mass spectrometry is a rapid method, which requires small sample amounts for either identification of protein-bound ligands or for determination of the apparent gas phase protein-ligand complex binding strengths.
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http://dx.doi.org/10.3390/molecules25204776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587528PMC
October 2020

ITEM-THREE analysis of a monoclonal anti-malaria antibody reveals its assembled epitope on the MSP1 antigen.

J Biol Chem 2020 10 26;295(44):14987-14997. Epub 2020 Aug 26.

Proteome Center Rostock, University Medicine Rostock and University of Rostock, Rostock, Germany. Electronic address:

Rapid diagnostic tests are first-line assays for diagnosing infectious diseases, such as malaria. To minimize false positive and false negative test results in population-screening assays, high-quality reagents and well-characterized antigens and antibodies are needed. An important property of antigen-antibody binding is recognition specificity, which best can be estimated by mapping an antibody's epitope on the respective antigen. We have cloned a malarial antigen-containing fusion protein, MBP-MSP1, in , which then was structurally and functionally characterized before and after high pressure-assisted enzymatic digestion. We then used our previously developed method, intact transition epitope mapping-targeted high-energy rupture of extracted epitopes (ITEM-THREE), to map the area on the MBP-MSP1 antigen surface that is recognized by the anti-MSP1 antibody G17.12. We identified three epitope-carrying peptides, GRNISQHQCVKKQCPQNSGCFRHLDE, GRNISQHQCVKKQCPQNSGCFRHLDEREE, and CKCLLNYKQE, from the GluC-derived peptide mixture. These peptides belong to an assembled (conformational) epitope on the MBP-MSP1 antigen whose identification was substantiated by positive and negative control experiments. In conclusion, our data help to establish a workflow to obtain high-quality control data for diagnostic assays, including the use of ITEM-THREE as a powerful analytical tool. Data are available via ProteomeXchange: PXD019717.
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http://dx.doi.org/10.1074/jbc.RA120.014802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606685PMC
October 2020

Intact Transition Epitope Mapping - Thermodynamic Weak-force Order (ITEM - TWO).

J Proteomics 2020 02 1;212:103572. Epub 2019 Nov 1.

Proteome Center Rostock, University Medicine Rostock, Rostock, Germany. Electronic address:

We have developed an electrospray mass spectrometry method which is capable to determine antibody affinity in a gas phase experiment. A solution with the immune complex is electrosprayed and multiply charged ions are translated into the gas phase. Then, the intact immune-complex ions are separated from unbound peptide ions. Increasing the voltage difference in a collision cell results in collision induced dissociation of the immune-complex by which bound peptide ions are released. When analyzing a peptide mixture, measuring the mass of the complex-released peptide ions identifies which of the peptides contains the epitope. A step-wise increase in the collision cell voltage difference changes the intensity ratios of the surviving immune complex ions, the released peptide ions, and the antibody ions. From all the ions´ normalized intensity ratios are deduced the thermodynamic quasi equilibrium dissociation constants (K) from which are calculated the apparent gas phase Gibbs energies of activation over temperature (ΔGT). The order of the apparent gas phase dissociation constants of four antibody - epitope peptide pairs matched well with those obtained from in-solution measurements. The determined gas phase values for antibody affinities are independent from the source of the investigated peptides and from the applied instrument. Data are available via ProteomeXchange with identifier PXD016024. SIGNIFICANCE: ITEM - TWO enables rapid epitope mapping and determination of apparent dissociation energies of immune complexes with minimal in-solution handling. Mixing of antibody and antigen peptide solutions initiates immune complex formation in solution. Epitope binding strengths are determined in the gas phase after electrospraying the antibody / antigen peptide mixtures and mass spectrometric analysis of immune complexes under different collision induced dissociation conditions. Since the order of binding strengths in the gas phase is the same as that in solution, ITEM - TWO characterizes two most important antibody properties, specificity and affinity.
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http://dx.doi.org/10.1016/j.jprot.2019.103572DOI Listing
February 2020

Intact Transition Epitope Mapping - Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE).

Mol Cell Proteomics 2019 08 30;18(8):1543-1555. Epub 2019 May 30.

‡Proteome Center Rostock, University Medicine Rostock, Rostock, Germany. Electronic address:

Epitope mapping, which is the identification of antigenic determinants, is essential for the design of novel antibody-based therapeutics and diagnostic tools. ITEM-THREE is a mass spectrometry-based epitope mapping method that can identify epitopes on antigens upon generating an immune complex in electrospray-compatible solutions by adding an antibody of interest to a mixture of peptides from which at least one holds the antibody's epitope. This mixture is nano-electrosprayed without purification. Identification of the epitope peptide is performed within a mass spectrometer that provides an ion mobility cell sandwiched in-between two collision cells and where this ion manipulation setup is flanked by a quadrupole mass analyzer on one side and a time-of-flight mass analyzer on the other side. In a stepwise fashion, immune-complex ions are separated from unbound peptide ions and dissociated to release epitope peptide ions. Immune complex-released peptide ions are separated from antibody ions and fragmented by collision induced dissociation. Epitope-containing peptide fragment ions are recorded, and mass lists are submitted to unsupervised data base search thereby retrieving both, the amino acid sequence of the epitope peptide and the originating antigen. ITEM-THREE was developed with antiTRIM21 and antiRA33 antibodies for which the epitopes were known, subjecting them to mixtures of synthetic peptides of which one contained the respective epitope. ITEM-THREE was then successfully tested with an enzymatic digest of His-tagged recombinant human β-actin and an antiHis-tag antibody, as well as with an enzymatic digest of recombinant human TNFα and an antiTNFα antibody whose epitope was previously unknown.
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http://dx.doi.org/10.1074/mcp.RA119.001429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683010PMC
August 2019

Mass spectrometric characterization of protein structures and protein complexes in condensed and gas phase.

Eur J Mass Spectrom (Chichester) 2017 Dec 26;23(6):445-459. Epub 2017 Jul 26.

1 Proteome Center Rostock, University of Rostock, Rostock, Germany.

Proteins are essential for almost all physiological processes of life. They serve a myriad of functions which are as varied as their unique amino acid sequences and their corresponding three-dimensional structures. To fulfill their tasks, most proteins depend on stable physical associations, in the form of protein complexes that evolved between themselves and other proteins. In solution (condensed phase), proteins and/or protein complexes are in constant energy exchange with the surrounding solvent. Albeit methods to describe in-solution thermodynamic properties of proteins and of protein complexes are well established and broadly applied, they do not provide a broad enough access to life-science experimentalists to study all their proteins' properties at leisure. This leaves great desire to add novel methods to the analytical biochemist's toolbox. The development of electrospray ionization created the opportunity to characterize protein higher order structures and protein complexes rather elegantly by simultaneously lessening the need of sophisticated sample preparation steps. Electrospray mass spectrometry enabled us to translate proteins and protein complexes very efficiently into the gas phase under mild conditions, retaining both, intact protein complexes, and gross protein structures upon phase transition. Moreover, in the environment of the mass spectrometer (gas phase, in vacuo), analyte molecules are free of interactions with surrounding solvent molecules and, therefore, the energy of inter- and intramolecular forces can be studied independently from interference of the solvating environment. Provided that gas phase methods can give information which is relevant for understanding in-solution processes, gas phase protein structure studies and/or investigations on the characterization of protein complexes has rapidly gained more and more attention from the bioanalytical scientific community. Recent reports have shown that electrospray mass spectrometry provides direct access to six prime protein complex properties: stabilities, compositions, binding surfaces (epitopes), disassembly processes, stoichiometries, and thermodynamic parameters.
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http://dx.doi.org/10.1177/1469066717722256DOI Listing
December 2017

Apparent activation energies of protein-protein complex dissociation in the gas-phase determined by electrospray mass spectrometry.

Anal Bioanal Chem 2017 Nov 12;409(28):6549-6558. Epub 2017 Sep 12.

Proteome Center Rostock, University Rostock Medical Center, Schillingallee 69, 18059, Rostock, Germany.

We have developed a method to determine apparent activation energies of dissociation for ionized protein-protein complexes in the gas phase using electrospray ionization mass spectrometry following the Rice-Ramsperger-Kassel-Marcus quasi-equilibrium theory. Protein-protein complexes were formed in solution, transferred into the gas phase, and separated from excess free protein by ion mobility filtering. Afterwards, complex disassembly was initiated by collision-induced dissociation with step-wise increasing energies. Relative intensities of ion signals were used to calculate apparent activation energies of dissociation in the gas phase by applying linear free energy relations. The method was developed using streptavidin tetramers. Experimentally determined apparent gas-phase activation energies for dissociation ([Formula: see text]) of complexes consisting of Fc parts from immunoglobulins (IgG-Fc) and three closely related protein G' variants (IgG-Fc•protein G'e, IgG-Fc•protein G'f, and IgG-Fc•protein G'g) show the same order of stabilities as can be inferred from their in-solution binding constants. Differences in stabilities between the protein-protein complexes correspond to single amino acid residue exchanges in the IgG-binding regions of the protein G' variants. Graphical abstract Electrospray mass spectrometry and collision-induced dissociation delivers apparent activation energies and supramolecular bond force constants of protein-protein complexes in the gas phase.
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http://dx.doi.org/10.1007/s00216-017-0603-4DOI Listing
November 2017

Mass spectrometric epitope mapping.

Mass Spectrom Rev 2018 03 12;37(2):229-241. Epub 2016 Jul 12.

Proteome Center Rostock, University Medicine and Natural Science Faculty, University of Rostock, Rostock, Germany.

Mass spectrometric epitope mapping has become a versatile method to precisely determine a soluble antigen's partial structure that directly interacts with an antibody in solution. Typical lengths of investigated antigens have increased up to several 100 amino acids while experimentally determined epitope peptides have decreased in length to on average 10-15 amino acids. Since the early 1990s more and more sophisticated methods have been developed and have forwarded a bouquet of suitable approaches for epitope mapping with immobilized, temporarily immobilized, and free-floating antibodies. While up to now monoclonal antibodies have been mostly used in epitope mapping experiments, the applicability of polyclonal antibodies has been proven. The antibody's resistance towards enzymatic proteolysis has been of key importance for the two mostly applied methods: epitope excision and epitope extraction. Sample consumption has dropped to low pmol amounts on both, the antigen and the antibody. While adequate in-solution sample handling has been most important for successful epitope mapping, mass spectrometric analysis has been found the most suitable read-out method from early on. The rapidity by which mass spectrometric epitope mapping nowadays is executed outperforms all alternative methods. Thus, it can be asserted that mass spectrometric epitope mapping has reached a state of maturity, which allows it to be used in any mass spectrometry laboratory. After 25 years of constant and steady improvements, its application to clinical samples, for example, for patient characterization and stratification, is anticipated in the near future. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:229-241, 2018.
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http://dx.doi.org/10.1002/mas.21516DOI Listing
March 2018

A Dynamic Model of pH-Induced Protein G'e Higher Order Structure Changes derived from Mass Spectrometric Analyses.

Anal Chem 2016 Jan 9;88(1):890-7. Epub 2015 Dec 9.

Proteome Center Rostock, University Medicine Rostock , Schillingallee 69, 18059 Rostock, Germany.

To obtain insight into pH change-driven molecular dynamics, we studied the higher order structure changes of protein G'e at the molecular and amino acid residue levels in solution by using nanoESI- and IM-mass spectrometry, CD spectroscopy, and protein chemical modification reactions (protein footprinting). We found a dramatic change of the overall tertiary structure of protein G'e when the pH was changed from neutral to acidic, whereas its secondary structure features remained nearly invariable. Limited proteolysis and surface-topology mapping of protein G'e by fast photochemical oxidation of proteins (FPOP) under neutral and acidic conditions reveal areas where higher order conformational changes occur on the amino-acid residue level. Under neutral solution conditions, lower oxidation occurs for residues of the first linker region, whereas greater oxidative modifications occur for amino-acid residues of the IgG-binding domains I and II. We propose a dynamic model of pH-induced structural changes in which protein G'e at neutral pH adopts an overall tight conformation with all four domains packed in a firm assembly, whereas at acidic pH, the three IgG-binding domains form an elongated alignment, and the N-terminal, His-tag-carrying domain unfolds. At the same time the individual IgG-binding domains themselves seem to adopt a more compacted fold. As the secondary structure features are nearly unchanged at either pH, interchange between both conformations is highly reversible, explaining the high reconditioning power of protein G'e-based affinity chromatography columns.
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http://dx.doi.org/10.1021/acs.analchem.5b03536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201196PMC
January 2016

Ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for phosphopeptide analysis with a solidified ionic liquid matrix.

Eur J Mass Spectrom (Chichester) 2015 ;21(2):65-77

Proteome Center Rostock, University of Rostock, Germany..

A solidified ionic liquid matrix (SILM) consisting of 3-aminoquinoline, α-cyano-4- hydroxycinnamic acid and ammonium dihydrogen phosphate combines the benefits of liquid and solid MALDI matrices and proves to be well suitable for phosphopeptide analysis using MALDI-MS in the low femtomole range. Desalting and buffer exchange that typically follow after phosphopeptide elution from metal oxide affinity chromatography (MOAC) materials can be omitted. Shifting the pH from acidic to basic during target preparation causes slow matrix crystallization and homogeneous embedding of the analyte molecules, forming a uniform preparation from which (phospho)peptides can be ionized in high yields over long periods of time. The novel combination of MOAC-based phosphopeptide enrichment with SILM preparation has been developed with commercially available standard phosphopeptides and with α-casein as phosphorylated standard protein. The applicability of the streamlined phosphopeptide analysis procedure to cell biological and clinical samples has been tested (i) using affinity-enriched endogenous TRIM28 from cell cultures and (ii) by analysis of a two-dimensional gel-separated protein spot from a bladder cancer sample.
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http://dx.doi.org/10.1255/ejms.1362DOI Listing
September 2015

Mass spectrometric characterization of limited proteolysis activity in human plasma samples under mild acidic conditions.

Methods 2015 Nov 26;89:30-7. Epub 2015 Feb 26.

Proteome Center Rostock, University Medicine Rostock, Rostock, Germany. Electronic address:

We developed a limited proteolysis assay for estimating dynamics in plasma-borne protease activities using MALDI ToF MS analysis as readout. A highly specific limited proteolysis activity was elicited in human plasma by shifting the pH to 6. Mass spectrometry showed that two singly charged ion signals at m/z 2753.44 and m/z 2937.56 significantly increased in abundance under mild acidic conditions as a function of incubation time. For proving that a provoked proteolytic activity in mild acidic solution caused the appearance of the observed peptides, control measurements were performed (i) with pepstatin as protease inhibitor, (ii) with heat-denatured samples, (iii) at pH 1.7, and (iv) at pH 7.5. Mass spectrometric fragmentation analysis showed that the observed peptides encompass the amino acid sequences 1-24 and 1-26 from the N-terminus of human serum albumin. Investigations on peptidase specificities suggest that the two best candidates for the observed serum albumin cleavages are cathepsin D and E. Reproducibility, robustness, and sensitivity prove the potential of the developed limited proteolysis assay to become of clinical importance for estimating dynamics of plasma-borne proteases with respect to associated pathophysiological tissue conditions.
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http://dx.doi.org/10.1016/j.ymeth.2015.02.013DOI Listing
November 2015

"De-novo" amino acid sequence elucidation of protein G'e by combined "top-down" and "bottom-up" mass spectrometry.

J Am Soc Mass Spectrom 2015 Mar 6;26(3):482-92. Epub 2015 Jan 6.

Proteome Center Rostock, University Rostock Medical Center, Rostock, Germany.

Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G´ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant (K(d)) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins.
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http://dx.doi.org/10.1007/s13361-014-1053-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130978PMC
March 2015

A novel strategy for the rapid preparation and isolation of intact immune complexes from peptide mixtures.

J Mol Recognit 2014 Sep;27(9):566-74

Proteome Center Rostock, University Medicine Rostock, Rostock, Germany.

The development and application of a miniaturized affinity system for the preparation and release of intact immune complexes are demonstrated. Antibodies were reversibly affinity-adsorbed on pipette tips containing protein G´ and protein A, respectively. Antigen proteins were digested with proteases and peptide mixtures were exposed to attached antibodies; forming antibody-epitope complexes, that is, immune complexes. Elution with millimolar indole propionic acid (IPA)-containing buffers under neutral pH conditions allowed to effectively isolate the intact immune complexes in purified form. Size exclusion chromatography was performed to determine the integrity of the antibody-epitope complexes. Mass spectrometric analysis identified the epitope peptides in the respective SEC fractions. His-tag-containing recombinant human glucose-6-phosphate isomerase in combination with an anti-His-tag monoclonal antibody was instrumental to develop the method. Application was extended to the isolation of the intact antibody-epitope complex of a recombinant human tripartite motif 21 (rhTRIM21) auto-antigen in combination with a rabbit polyclonal anti-TRIM21 antibody. Peptide chip analysis showed that antibody-epitope binding of rhTRIM21 peptide antibody complexes was not affected by the presence of IPA in the elution buffer. By contrast, protein G´ showed an ion charge structure by electrospray mass spectrometry that resembled a denatured conformation when exposed to IPA-containing buffers. The advantages of this novel isolation strategy are low sample consumption and short experimental duration in addition to the direct and robust methodology that provides easy access to intact antibody-antigen complexes under neutral pH and low salt conditions for subsequent investigations.
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http://dx.doi.org/10.1002/jmr.2375DOI Listing
September 2014

Mass spectrometric profiling of cord blood serum proteomes to distinguish infants with intrauterine growth restriction from those who are small for gestational age and from control individuals.

Transl Res 2014 Jul 10;164(1):57-69. Epub 2013 Dec 10.

Proteome Center Rostock, Medical Faculty and Natural Science Faculty, University of Rostock, Rostock, Germany. Electronic address:

Intrauterine growth restriction (IUGR) is a multifactorial condition in that the fetus does not reach its genetically given growth potential. Besides its contribution to perinatal mortality, it is a risk factor for cardiovascular and metabolic diseases later in life. The diagnosis is based on antenatal sonography, which allows differentiating between IUGR and fetuses that are small by constitution (small for gestational age [SGA]). Yet, neither a clinical nor a biochemical tool is available to confirm reliably the diagnosis of IUGR postnatally. Recently, we identified umbilical cord blood proteins of the apolipoprotein family by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with differential signal intensities between the IUGR group and a control group. We hypothesized that identified molecules have the potential to generate a proteome profile specific for IUGR. A total of 114 serum samples (42 from the IUGR group, 12 from the SGA group, and 60 from the control group) of the umbilical vein (99 samples) and umbilical artery (15 samples) were analyzed. Sample quality was estimated by determining the abundance of hemoglobin (hemolysis) and CXC-motif chemokines CXCL4 and CXCL7 (platelet activation). Samples passing the quality criteria were forwarded to multiplex apolipoprotein profiling. Assay performance was tested with the sample sets, resulting in a sensitivity of 0.91 and a specificity of 0.85 in the test set with venous blood samples. Arterial cord blood samples followed the trend (sensitivity, 0.67; specificity, 0.85). SGA samples grouped together with the control samples. We conclude that the proteome profiling signature is confirmatory to clinical surveillance with the potential to identify neonates with IUGR postnatally in low-birth weight babies born at uncertain gestational age when antenatal sonography data have not been recorded.
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http://dx.doi.org/10.1016/j.trsl.2013.12.003DOI Listing
July 2014

Molecular adaptations of Helicoverpa armigera midgut tissue under pyrethroid insecticide stress characterized by differential proteome analysis and enzyme activity assays.

Comp Biochem Physiol Part D Genomics Proteomics 2013 Jun 24;8(2):152-62. Epub 2013 Apr 24.

Biochemistry Joint Graduate Program, Middle East Technical University, Ankara, Turkey.

Helicoverpa armigera is an insect that causes important economic losses in crops. To reduce this loss, pyrethroids have been commonly used against H. armigera in farming areas. However, excess and continuous usage of pyrethroids cause resistance in H. armigera. Therefore, expressions of midgut proteins of two H. armigera field populations were compared to those of a susceptible strain by 2-D PAGE and MALDI-ToF-MS. Our results indicate that H. armigera reacts to pyrethroid-induced stress mainly by increasing the expression of energy metabolism-related proteins, such as ATP synthase and arginine kinase. NADPH cytochrome P450 reductase, also up-regulated, could play a role in detoxification of toxic pyrethroid metabolites, such as 3-phenoxybenzaldehyde. Interestingly, while GSTs were not found up-regulated in the comparative proteome analysis, biochemical assays showed significant increases of enzyme activities in both field populations as compared to the susceptible strain. Similarly, although esterases were not found differentially expressed, biochemical assays showed significant increases of esterase activities in both field populations. Thus, esterases are also proposed to be involved in metabolic responses towards pyrethroid insecticide-induced stress. In conclusion, we suggest increased energy metabolism in the midgut tissue of H. armigera as a general prerequisite for compensating the costs of energy-consuming detoxification processes.
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http://dx.doi.org/10.1016/j.cbd.2013.04.001DOI Listing
June 2013

A proteome signature for intrauterine growth restriction derived from multifactorial analysis of mass spectrometry-based cord blood serum profiling.

Electrophoresis 2012 Jul;33(12):1881-93

Proteome Center Rostock, Medical Faculty and Natural Science Faculty, University of Rostock, Rostock, Germany.

Intrauterine growth restriction (IUGR) is defined as a condition in which the fetus does not reach its genetically given growth potential, resulting in low birth weight. IUGR is an important cause of perinatal morbidity and mortality, thus contributing substantially to medically indicated preterm birth in order to prevent fetal death. We subjected umbilical cord blood serum samples either belonging to the IUGR group (n = 15) or to the control group (n = 15) to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were analyzed by MALDI-TOF mass spectrometric profiling. The six best differentiating ion signals at m/z 8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z 16 001 were collectively assigned as IUGR proteome signature. Separation confidence of our IUGR proteome signature reached a sensitivity of 0.87 and a specificity of 0.93. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS-PAGE in conjunction with peptide mass fingerprint analysis of cord blood serum proteins. One constituent of this proteome signature, apolipoprotein C-III(0) , a derivative lacking glycosylation, has been found more abundant in the IUGR cord blood serum samples, irrespective of gestational age. Hence, we suggest apolipoprotein C-III(0) as potential key-marker of the here proposed IUGR proteome signature, as it is a very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) member and as such involved in triglyceride metabolism that itself is discussed as being of importance in IUGR pathogenesis. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.
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http://dx.doi.org/10.1002/elps.201200001DOI Listing
July 2012

Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: a pilot study with colorectal carcinoma patient samples.

Anal Bioanal Chem 2012 Apr 16;403(1):227-38. Epub 2012 Feb 16.

Department of General Surgery, Molecular Oncology and Immunotherapy, Medical Faculty, University of Rostock, Rostock, Germany.

Patient-specific sequential epitopes were identified by peptide chip analysis using 15mer peptides immobilized on glass slides that covered the topoisomerase IIa protein with a frameshift of five amino acids. Binding specificities of serum antibodies against sequential epitopes were confirmed as being mono-specific by peptide chip re-analysis of epitope-affinity-purified antibody pools. These results demonstrate that serum samples from colon carcinoma patients contain antibodies against sequential epitopes from the topoisomerase IIa antigen. Interactions of patients' antibodies with sequential epitopes displayed by peptides on glass surfaces may thus mirror disease-specific immune situations. Consequently, these data suggest epitope-antibody reactivities on peptide chips as potential diagnostic readouts of individual immune response characteristics, especially because monospecific antibodies can be interrogated. Subsequently, the clonality of the antibodies present in the mono-specific antibody pools was characterized by 2D gel electrophoresis. This analysis suggested that the affinity-purified antibodies were oligoclonal. Similarly to large-scale screening approaches for specific antigen-antibody interactions in order to improve disease diagnostic, we suggest that "protein-wide" screening for specific epitope-paratope interactions may help to develop novel assays for monitoring of personalized therapies, since individual properties of antigen-antibody interactions remain distinguishable.
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http://dx.doi.org/10.1007/s00216-012-5781-5DOI Listing
April 2012

Toponostics of invasive ductal breast carcinoma: combination of spatial protein expression imaging and quantitative proteome signature analysis.

Int J Clin Exp Pathol 2011 Mar 28;4(5):454-67. Epub 2011 Feb 28.

Proteome Center Rostock, University of Rostock, Rostock, Germany.

Due to enormous advances in quantitative proteomics and in immunohistochemistry (pathology), the two research areas have now reached the state to be successfully interwoven in order to tackle challenges in toponostics and to open tumor-targeted systems pathology approaches. In this study the differential expressions of candidate proteins nucleophosmin, nucleoside diphosphate kinase A/B (NDKA/B), osteoinducive factor (mimecan), and pyru-vate kinase M2 from a quantitative proteome signature for invasive ductal breast cancer were determined by immunohistochemistry on 53 tissue slices from formalin-fixed and paraffin-embedded tumor and control tissue samples from ten patients and fourteen controls. In addition, 87 images from the Human Protein Atlas representing seven tumor and nine normal breast tissue samples were investigated by computer-assisted semi-quantitative density measurements on nucleophosmin, nucleoside diphosphate kinase A/B (NDKA/B), osteoinducive factor (mimecan), pyruvate kinase M2, glyceraldehyde-3-phosphate dehydro-genase (GAP-DH), and mimecan (osteoinductive factor). Both IHC data sets match well to each other and support the quantitative proteome analysis data. Determining spatial distribution of signature protein expressions by protein imaging on morphologically intact tissue samples at the sub-cellular level and, hence, keeping all topological information, presents an added value to quantitative proteome data. Such comprehensive data sets are needed for both, pathway analyses and for "next generation clinical diagnostics" approaches.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3127067PMC
March 2011

Mass spectrometric characterization of protein structure details refines the proteome signature for invasive ductal breast carcinoma.

J Am Soc Mass Spectrom 2011 Mar 8;22(3):440-56. Epub 2011 Feb 8.

Proteome Center Rostock, Department for Proteome Research, Institute of Immunology, Medical Faculty, University of Rostock, Schillingallee 69, P.O. Box 100 888, Rostock 18055, Germany.

Early diagnosis as well as individualized therapies are necessary to reduce the mortality of breast cancer, and personalized patient care strategies rely on novel prognostic or predictive factors. In this study, with six breast cancer patients, 2D gel analysis was applied for studying protein expression differences in order to distinguish invasive ductal breast carcinoma, the most frequent breast tumor subtype, from control samples. In total, 1203 protein spots were assembled in a 2D reference gel. Differentially abundant spots were subjected to peptide mass fingerprinting for protein identification. Twenty proteins with their corresponding 38 differentially expressed 2D gel spots were contained in our previously reported proteome signature, suggesting that distinct protein forms were contributing. In-depth MS/MS measurements enabled analyses of protein structure details of selected proteins. In protein spots that significantly contributed to our signature, we found that glyceraldehyde-3-phosphate dehydrogenase was N-terminally truncated, pyruvate kinase M2 and nucleoside diphosphate kinase A but not other isoforms of these proteins were of importance, and nucleophosmin phosphorylation at serine residues 106 and 125 were clearly identified. Principle component analysis and hierarchical clustering with normalized quantitative data from the 38 spots resulted in accurate separation of tumor from control samples. Thus, separation of tissue samples as in our initial proteome signature could be confirmed even with a different proteome analysis platform. In addition, detailed protein structure investigations enabled refining our proteome signature for invasive ductal breast carcinoma, opening the way to structure-/function studies with respect to disease processes and/or therapeutic intervention.
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http://dx.doi.org/10.1007/s13361-010-0031-6DOI Listing
March 2011

Altered sialylation on the cell-surface proteins of dexamethasone-treated human macrophages contributes to augmented uptake of apoptotic neutrophils.

Immunol Lett 2011 Mar 16;135(1-2):88-95. Epub 2010 Oct 16.

Apoptosis and Genomics Research Group of the Hungarian Academy of Sciences, Research Centre for Molecular Medicine, Medical Faculty, University of Debrecen, Nagyerdei krt. 98, H-4032 Debrecen, Hungary.

Macrophages eliminate apoptotic granulocytes before their secondary necrosis during resolution of inflammation. A well-known glucocorticoid, the anti-inflammatory dexamethasone augments phagocytosis capacity of macrophages with a so far not fully clarified mechanism. We have found that sialylation of cell-surface proteins on human macrophages is markedly altered by dexamethasone. Compared to non-treated cells, dexamethasone-treated macrophages can bind significantly less Sambucus nigra lectin specific for sialic acids on their surfaces as a result of undersialylation of annexin-II and an HLA-II protein. Non-treated macrophages covered by S. nigra lectin had increased uptake of apoptotic cells; however, the significantly higher phagocytosis capacity of dexamethasone-treated macrophages could not be stimulated further this way. Our results suggest that dexamethasone treatment leads to decreased number of sialic acids on the surfaces of human macrophages promoting recognition and uptake of apoptotic cells.
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http://dx.doi.org/10.1016/j.imlet.2010.10.002DOI Listing
March 2011

Towards a proteome signature for invasive ductal breast carcinoma derived from label-free nanoscale LC-MS protein expression profiling of tumorous and glandular tissue.

Anal Bioanal Chem 2009 Dec 29;395(8):2443-56. Epub 2009 Oct 29.

Proteome Center Rostock, University of Rostock, 18055, Rostock, Germany.

As more and more alternative treatments become available for breast carcinoma, there is a need to stratify patients and individual molecular information seems to be suitable for this purpose. In this study, we applied label-free protein quantitation by nanoscale LC-MS and investigated whether this approach could be used for defining a proteome signature for invasive ductal breast carcinoma. Tissue samples from healthy breast and tumor were collected from three patients. Protein identifications were based on LC-MS peptide fragmentation data which were obtained simultaneously to the quantitative information. Hereby, an invasive ductal breast carcinoma proteome signature was generated which contains 60 protein entries. The on-column concentrations for osteoinductive factor, vimentin, GAP-DH, and NDKA are provided as examples. These proteins represent distinctive gene ontology groups of differentially expressed proteins and are discussed as risk markers for primary tumor pathogenesis. The developed methodology has been found well applicable in a clinical environment in which standard operating procedures can be kept; a prerequisite for the definition of molecular parameter sets that shall be capable for stratification of patients.
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http://dx.doi.org/10.1007/s00216-009-3187-9DOI Listing
December 2009

MS characterization of apheresis samples from rheumatoid arthritis patients for the improvement of immunoadsorption therapy - a pilot study.

Proteomics Clin Appl 2009 Jul;3(7):797-809

Proteome Center Rostock, University of Rostock, Rostock, Germany.

Identification of proteins from apheresis samples was performed by both SDS-PAGE and 2-D gel separation of eluted proteins from staphylococcal protein A-based immunoadsorption columns (Prosorba(®) ) followed by MS peptide mass fingerprinting and MS/MS peptide sequencing on a MALDI QIT TOF mass spectrometer. MS/MS peptide sequencing was performed in conjunction with a micro reversed phase HPLC configured with an online MALDI plate-spotting device. Apheresis treatment had been performed in three patients with longstanding therapy refractory rheumatoid arthritis. 2-D gels displayed ca. 500 spots representing proteins that were eluted from the Prosorba(®) columns. From 54 gels, a total of 1256 protein spots had been picked and yielded in the identification of 56 non-redundant proteins without counting isoforms. Proteins from the eluates belong to five major groups comprising (i) immunoglobulins (IgG, IgA, IgM heavy and light chains; about 40% of the spots), (ii) proteins involved in coagulation, (iii) HDL/LDL-associated proteins, (iv) proteins from the complement system, and (v) acute phase proteins. MS analysis showed that the full-length C3 complement protein had been cleaved upon complement activation, presumably on the column, such that the anaphylatoxin C3a was produced and released during therapy. Our results are consistent with clinical observations on both patient responses to therapy and reported adverse events. For the first time, direct molecular information has become available to support mechanistic reasoning for the principle of function of staphylococcal protein A-based immunoadsorption therapy and for the explanation of adverse events. According to our results, removal and/or modulation of immune complexes together with complement activation can be regarded as the major events that are taking place during Prosorba(®) therapy. In order to avoid complement activation and induction of an inflammatory cascade, we suggest the prevention of C3a anaphylatoxin-related reactions during immunoadsorption therapy.
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http://dx.doi.org/10.1002/prca.200800232DOI Listing
July 2009

Mass spectrometric proteome analysis suggests anaerobic shift in metabolism of Dauer larvae of Caenorhabditis elegans.

Biochim Biophys Acta 2008 Nov 14;1784(11):1763-70. Epub 2008 Jun 14.

Proteome Center Rostock, University of Rostock, Schillingallee 69, D-18057 Rostock, Germany.

The Dauer larva is a non-feeding alternative larval stage of some nematodes specialized for long-term survival and dispersal. In this study we compared proteome maps obtained from Dauer larvae with those from the corresponding third larval stage (L3) of the feeding life cycle of C. elegans wild-type strain N2. We demonstrate at the protein level that altered metabolism may participate in longevity determination of Dauers. We detected huge amounts of alcohol dehydrogenase (CE12212) and aldehyde dehydrogenase (CE29809) in Dauer animals, indicating highly active fermentative pathways. Inorganic pyrophosphatase (CE05448) that enables to metabolize pyrophosphate as a high-energy source was over-expressed in Dauers. An interesting differentially expressed protein was phosphatidylethanolamine-binding protein (CE38516) that was found in high abundance in samples from Dauer larvae. Protein synthesis may be lowered in Dauer animals by the reduced expression of splicing factor rsp-3 (CE31089) and methionyl-tRNA synthase (CE34219). We observed significantly lower amounts of the pepsin-like aspartyl protease 1 (CE21681) in non-feeding Dauers, which is in agreement with reduced nutrient digestion. Finally, the hypothetical protein R08E5.2 (CE33294) was present in high abundance in L3 animals.
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http://dx.doi.org/10.1016/j.bbapap.2008.05.017DOI Listing
November 2008

Cryodetector mass spectrometry profiling of plasma samples for HELLP diagnosis: an exploratory study.

Proteomics 2005 Aug;5(12):3079-87

Proteome Center Rostock, University of Rostock, Joachim-Jungius-Strasse 9, D-10859 Rostock, Germany.

The cryodetector MS-based screening method enables the rapid and reliable separation of hemolysis, elevated liver enzymes, and low platelets syndrome (HELLP) patients from healthy women. For developing the assay, plasma protein abundances from patients suffering from HELLP were profiled before and after delivery and compared to healthy pregnant and nonpregnant control individuals, using a TOF mass spectrometer equipped with a cryodetector system. The spectra were well reproducible when acquisition conditions were kept constant and the overall appearance of the profiles was well comparable. Peak areas of ten selected ion signals (9.5, 11.8, 14.0, 28.1, 43.4, 50.5, 60.1, 66.6, 74.5, and 79.8 kDa) from each spectrum were subjected to statistical analysis. Significant differences in ion intensities between the groups could be measured even without sample fractionation. The most striking difference between most of the spectra from HELLP patients and nonHELLP samples was the presence/absence of the 11.8 kDa ion signal (p-value: 0.0000365), most likely belonging to serum amyloid A (SAA). Other significant differences in ion intensities between HELLP and control samples were observed in the peak areas of the 14.0, 28.1, 50.5, and 74.5 kDa ion signals. Using the cryodetector MS data for sorting plasma samples into either the HELLP group (n = 8) or the nonHELLP group (n = 22), a sensitivity of 87.5% and a specificity of 100% were achieved. The positive predictive value of our screening assay was 100% and a negative predictive value of 95.6% was obtained with the samples included in this study.
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http://dx.doi.org/10.1002/pmic.200402098DOI Listing
August 2005

Mass spectrometric protein structure characterization reveals cause of migration differences of haptoglobin alpha chains in two-dimensional gel electrophoresis.

Proteomics 2004 Dec;4(12):3921-32

Proteome Center Rostock, University of Rostock, Germany.

Haptoglobin belongs to the major constituents of plasma and acts as hemoglobin-binding and acute-phase protein. Due to the occurrence of three major allelic variants and further structural modifications, the alpha chains of haptoglobin form varying spot patterns in two-dimensional gel electrophoresis (2-DE) gels, which is generally observed in differential proteome analyses using plasma or related body fluids of humans. In the present study plasma samples from 10 donors of initially unknown haptoglobin phenotype were separated by 2-DE and tryptic digests of excised haptoglobin alpha chain spots were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and MALDI-quadrupole ion trap TOF-MS. Haptoglobin alpha1S, alpha1F, as well as alpha2 chains were found to occur each with at least three structurally differing protein species: (i) the unmodified form, which corresponds to the sequence database entries; (ii) derivatives, in which asparagine at position five is deamidated to aspartic acid; and (iii) derivatives with an additional C-terminal arginine residue. These structural variants account for the most commonly observed spot patterns of haptoglobin alpha chains in Coomassie-stained gels. Additionally, a minor derivative of the haptoglobin alpha2 chain carrying both modifications, deamidation at position five and the C-terminal arginine residue, was identified. Theoretical pI values of the characterized structural variants are, consistent with their observed migration in the 2-DE gels.
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http://dx.doi.org/10.1002/pmic.200400825DOI Listing
December 2004

Primary structure details of haptoglobin alpha chain proteins from human plasma samples are resolved by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight multiple-stage tandem mass spectrometry sequencing.

Eur J Mass Spectrom (Chichester) 2004 ;10(3):393-9

Proteome Center Rostock, University of Rostock, Joachim-Jungius-Str. 9, D-18059 Rostock, Germany.

MALDI QIT ToF MS(n) analyses lead to the rapid identification of protein structural details, as readily interpretable spectra after peptide fragmentations were obtained showing ion signals with high abundance even with sample amounts in the low femtomole range. In our studies we show that the Hp alpha 1F form that contained a C-terminal arginine residue was found to be the only contributing component to spot 149. By contrast, spots 77 and 79 were found to consist of two haptoglobin forms each. Spot 77 consists of Hp alpha 1S and deamidated Hp alpha 1F, whereas spot 79 consists of Hp alpha 1F and of Hp alpha 1S that contains a C- terminal arginine residue. The use of ion traps, enabling the acquisition of MSn spectra serves as a powerful peptide sequencing method for the analysis of both, genetic differences and post-translational modification events as the main reason for the observed spot pattern in the 2-D gels of haptoglobin proteins.
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http://dx.doi.org/10.1255/ejms.610DOI Listing
July 2004

Matrix-assisted laser desorption/ionization- quadrupole ion trap-time of flight mass spectrometry sequencing resolves structures of unidentified peptides obtained by in-gel tryptic digestion of haptoglobin derivatives from human plasma proteomes.

Proteomics 2003 Jun;3(6):851-8

Proteome Center Rostock, University of Rostock, Rostock, Germany.

Two-dimensional gel electrophoresis-separated and excised haptoglobin alpha2-chain protein spots were subjected to in-gel digestion with trypsin. Previously unassigned peptide ion signals observed in mass spectrometric fingerprinting experiments were sequenced using the matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF) mass spectrometer and showed that the haptoglobin alpha-chain derivative under study was cleaved by trypsin unspecifically. Abundant cleavages occurred C-terminal to histidine residues at H23, H28, and H87. In addition, mild acidic hydrolysis leading to cleavage after aspartic acid residues at D13 was observed. The uninterpreted tandem mass spectrometry (MS/MS) spectrum of the peptide with ion signal at 2620.19 was submitted to database search and yielded the identification of the corresponding peptide sequence comprising amino acids (aa) aa65-87 from the haptoglobin alpha-chain protein. Also, the presence of a mixture of two tryptic peptides (mass to charge ratio m/z 1708.8; aa40-54, and aa99-113, respectively), that is caused by a tiny sequence variation between the two repeats in the haptoglobin alpha2-chain protein was resolved by MS/MS fragmentation using the MALDI-QIT-TOF mass spectrometer instrument. Advantageous features such as (i) easy parent ion creation, (ii) minimal sample consumption, and (iii) real collision induced dissociation conditions, were combined successfully to determine the amino acid sequences of the previously unassigned peptides. Hence, the novel mass spectrometric sequencing method applied here has proven effective for identification of distinct molecular protein structures.
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http://dx.doi.org/10.1002/pmic.200300381DOI Listing
June 2003
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