Enzymatic diagnosis of neuronal lipofuscinoses in dried blood spots using substrates for concomitant tandem mass spectrometry and fluorimetry
2021-01, Maeser, Stefan, Petre, Brînduşa-Alina, Ion, Laura, Rawer, Stephan, Kohlschütter, Alfried, Santorelli, Filippo M, Simonati, Alessandro, Schulz, Angela, Przybylski, Michael
Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative diseases predominantly in childhood that are characterized by psychomotor deterioration, epilepsy, and early death of patients. The NCLs analyzed in the present study are caused by defects of the specific enzymes, CLN1 (palmitoyl protein thioesterase 1; PPT1), CLN2 (tripeptidyl peptidase 1; TPP1), and CLN10 (cathepsin D). Specific and sensitive diagnostic assays of NCLs were the main goal of this study. They are of increasing importance, particularly since enzyme replacement therapy (ERT) for NCL2 has recently become available for clinical treatment, and ERTs for further NCLs are under development. Here, we report specific and sensitive determinations for CLN1, CLN2, and CLN10 on dried blood spots by tandem mass spectrometry using multiple reaction monitoring mass spectrometry (MRM-MS). Identical substrates suitable for (i) fluorimetric determination of single enzymes and (ii) for MRM-MS determination of multiple enzymes were synthesized by chemical coupling of alkyl-umbelliferone building blocks with the corresponding peptidyl-substrate groups recognized by the target enzyme. Enzymatic determinations were performed both by fluorimetry and MRM-MS in patients with NCL1, NCL2, and NCL10 and showed good agreement in single assays. Moreover, duplex and triplex determinations were successfully performed for NCL1, NCL2, and NCL10. Specific peptidyl-(4-alkyl-umbelliferone) substrates were also synthesized for mass spectrometric determinations of different cathepsins (cathepsins-D, -F, and -B), to provide a differentiation of proteolytic specificities.
Epitope motif of an anti-nitrotyrosine antibody specific for tyrosine-nitrated peptides revealed by a combination of affinity approaches and mass spectrometry
2011-01, Drăguşanu, Mihaela, Petre, Brînduşa-Alina, Przybylski, Michael
Nitration of tyrosine residues has been shown to be an important oxidativemodification in proteins and has been suggested to AQ1 play a role in several diseases such as atherosclerosis, asthma, and lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine-specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified byMS.Wehave used amonoclonal 3-NT-specific antibody, and have synthesized a series of tyrosine-nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr-430 had been previously identified upon reaction with peroxynitrite . The determination of antibody-binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr-430, Tyr-421, Tyr-83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N-terminal to the nitration site. The highest affinity to the anti-3NT-antibody was found for the PCS peptide comprising the Tyr-430 nitration site with a KD of 60 nM determined for the peptide, PCS(424-436-Tyr-430NO2); in contrast, PCS peptides nitrated at Tyr-421 and Tyr-83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody-bound peptides and affinity-MS analysis revealed highest affinity to the antibody for tyrosine-nitrated peptides that contained positively charged amino acids in the N-terminal sequence to the nitration site. Remarkably, similar N-terminal sequences of tyrosine-nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil-cationic protein.
Protein-peptide affinity determination using an H/D exchange dilution strategy : Application to antigen-antibody interactions
2010, Tu, Tingting, Drăguşanu, Mihaela, Petre, Brînduşa-Alina, Rempel, Don L., Przybylski, Michael, Gross, Michael L.
A new methodology using hydrogen/deuterium amide exchange (HDX) to determine the binding affinity of protein-peptide interactions is reported. The method, based on our previously established approach, protein ligand interaction by mass spectrometry, titration, and H/D exchange (PLIMSTEX) [J. Am. Chem. Soc.2003, 125, 5252–5253], makes use of a dilution strategy (dPLIMSTEX) for HDX, using the mass of the peptide ligand as readout. We employed dPLIMSTEX to study the interaction of calcium-saturated calmodulin with the opioid peptide β-endorphin as a model system; the affinity results are in good agreement with those from traditional PLIMSTEX and with literature values obtained by using other methods. We show that the dPLIMSTEX method is feasible to quantify an antigen-antibody interaction involving a 3-nitrotyrosine modified peptide in complex with a monoclonal anti-nitrotyrosine antibody. A dissociation constant in the low nanomolar range was determined, and a binding stoichiometry of antibody/peptide of 1:2 was confirmed. In addition, we determined that the epitope in the binding interface contains a minimum of five amino acids. The dPLIMSTEX approach is a sensitive and powerful tool for the quantitative determination of peptide affinities with antibodies, complementary to conventional immuno-analytical techniques.
Post-translational Tyrosine Nitration of Eosinophil Granule Toxins Mediated by Eosinophil Peroxidase
2008, Ulrich, Martina, Petre, Brînduşa-Alina, Youhnovski, Nikolay, Prömm, Franziska, Schirle, Markus, Schumm, Michael, Pero, Ralph S., Doyle, Alfred, Checkel, James, Kita, Hirohito, Thiyagarajan, Nethaji, Acharya, K. Ravi, Schmid-Grendelmeier, Peter, Simon, Hans-Uwe, Schwarz, Heinz, Tsutsui, Masato, Shimokawa, Hiroaki, Bellon, Gabriel, Lee, James J., Przybylski, Michael, Döring, Gerd
Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO-/-, gp91phox-/-, and NOS-/- mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.
Affinity-Mass Spectrometry Approaches for Elucidating Structures and Interactions of Protein-Ligand Complexes
2014, Petre, Brînduşa-Alina
Affinity-based approaches in combination with mass spectrometry for molecular structure identification in biological complexes such as protein-protein, and protein-carbohydrate complexes have become popular in recent years. Affinity-mass spectrometry involves immobilization of a biomolecule on a chemically activated support, affinity binding of ligand(s), dissociation of the complex, and mass spectrometric analysis of the bound fraction. In this chapter the affinity-mass spectrometric methodologies will be presented for (1) identification of the epitope structures in the Abeta amyloid peptide, (2) identification of oxidative modifications in proteins such as nitration of tyrosine, (3) determination of carbohydrate recognition domains, and as (4) development of a biosensor chip-based mass spectrometric system for concomitant quantification and identification of protein-ligand complexes.
Degradation and oxidation postmortem of myofibrillar proteins in porcine skeleton muscle revealed by high resolution mass spectrometric proteome analysis
2011, Bernevic, Bogdan, Petre, Brînduşa-Alina, Galetskiy, Dmitry, Werner, Carsten, Wicke, Michael, Schellander, Karl, Przybylski, Michael
Early postmortem changes of porcine muscle proteins including the rate and extent of pH decline, proteolysis and oxidation are key factors influencing the loss of water in meat, and proteolytic degradation may result in shrinking of muscle cells and drip loss. We report here the identification and structural characterisation of post-mortem degradation and oxidation of myofibrillar proteins using high resolution mass spectrometric proteomics. Soluble muscle proteins from M. Longissimus dorsi obtained 48 h postmortem at different drip loss were separated by two-dimensional gel electrophoresis (2D-PAGE), and degradation products were identified by Fourier-transform ion cyclotron resonance mass spectrometry. Oxidation products were detected by 2D-oxyblot analysis of 2,4-dinitrophenylhydrazine (DNPH)-treated proteins using an anti-DNP antibody, and selected spots were identified by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Postmortem denaturation at low drip loss was found for four contractile proteins, myosin-light chain-1; myosin regulatory light chain; α-β-tropomyosin and α-actin. The combination of 2D-PAGE and FTICR-MS was found to be a powerful approach for identification of muscle protein degradation products, providing identification of several truncation forms of creatine kinase and troponin T. The comparison of 2D-oxyblot and silver-stained 2D-gels at low and high drip loss revealed approximately 70 oxidatively modified proteins from muscle cell lysate. Oxidative modifications, representing possible biomarker candidates, were identified at Lys-170 of creatine kinase (4-hydroxynonenal), Lys-326 of actin (amino-adipic semialdehyde), and at W-169 (kynurenine) of triosephosphate isomerase.
Interaction of beta-amyloid(1-40) peptide with pairs of metal ions : an electrospray ion trap mass spectrometric model study
2009, Drochioiu, Gabi, Manea, Marilena, Drăguşanu, Mihaela, Murariu, Manuela, Dragan, Ecaterina Stela, Petre, Brînduşa-Alina, Mezö, Gábor, Przybylski, Michael
The stoichiometries and the affinity toward simple and paired metal ions of synthetic amyloid-β(1-40) peptide (Aβ1-40) were investigated by electrospray ion trap mass spectrometry (ESI-MS), circular dichroism (CD), and atomic force microscopy (AFM). The results lead to the working hypothesis that pH-dependent metal binding to Aβ1-40 may induce conformational changes, which affect the affinity toward other metals. A significant copper and zinc binding to Aβ1-40 peptide at pH 5.5 was found, whereas nickel ions commonly bind to each molecule of β-amyloid peptide. Some complexes of Aβ1-40 with more than one nickel ion were identified by ESI-MS. In addition, nickel ions proved to enhance Aβ oligomerization. On increasing pH, up to 12 ions of zinc may bind to a single Aβ molecule. Under the same pH and concentration conditions, the binding pattern of the independent copper and silver ions to Aβ1-40 was different from that of the equimolecular mixture of the two metal ions. One might assume that some conformational changes due to water loss altered the capacity of Aβ peptide to bind certain heavy metal ions. As a consequence, copper silver interaction with the binding process to Aβ1-40 became highly complex. A competition between silver and nickel ions for Aβ1-40 binding sites at high pH was also observed. New strategies were proposed to identify the characteristic signals for some important metal ion peptide complexes in the spectra recorded at high pH or high concentrations of metal ions. To explain the formation of such a large number of high metal ion Aβ complexes, we took into consideration the participation of both histidine residues and free amino groups as well as carboxylate ones in the binding process. Finally, CD and AFM studies supported the mass spectrometric data.
When is Mass Spectrometry combined with Affinity Approaches essential? : A case study of Tyrosine Nitration in Proteins
2012-11, Petre, Brînduşa-Alina, Ulrich, Martina, Stumbaum, Mihaela, Bernevic, Bogdan, Moise, Adrian, Döring, Gerd, Przybylski, Michael
Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer’s disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar KD values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.
On-line bioaffinity-electrospray mass spectrometry for simultaneous detection, identification, and quantification of protein-ligand interactions
2010, Drăguşanu, Mihaela, Petre, Brînduşa-Alina, Slamnoiu, Stefan, Vlad, Camelia, Tu, Tingting, Przybylski, Michael, Dragusanu, Mihaela
We describe here an on-line combination of a surface acoustic wave (SAW) biosensor with electrospray ionization mass spectrometry (SAW-ESI-MS) that enables the direct detection,identification, and quantification of affinity-bound ligands from a protein ligand complex on a biosensor chip. A trapping column was used between the SAW-biosensor and the electrospray mass spectrometer equipped with a micro-guard column, which provides simultaneous sample concentration and desalting for the mass spectrometric analysis of the dissociated ligand. First applications of the on-line SAW-ESI-MS combination include (1), differentiation of â-amyloid (Aâ) epitope peptides bound to anti-Aâ antibodies; (2), the identification of immobilized Substance P peptide calmodulin complex; (3), identification and quantification of the interaction of 3-nitrotyrosine-modified peptides with nitrotyrosine-specific antibodies; and (4), identification of immobilized anti-á-synuclein human á-synuclein complex. Quantitative determinations of protein ligand complexes by SAW yielded dissociation constants (KD) from micro-to low nanomolar sample concentrations. The on-line bioaffinity-ESI-MS combination presented here is expected to enable broad bioanalytical application to the simultaneous, label-free determination and quantification of biopolymer-ligand interactions, as diverse as antigen-antibody and lectin-carbohydrate complexes.
Molecular Recognition Specificity of anti-3-nitrotyrosine Antibodies Revealed by Affinity-Mass Spectrometry and Immunoanalytical Methods
2008, Petre, Brînduşa-Alina, Drăguşanu, Mihaela, Przybylski, Michael
Nitration of tyrosine residues in proteins has been mainly characterised by immunoanalytical methods using anti-3-nitrotyrosine antibodies, and nitration sites and sequences have been hitherto identified only in a few cases using mass spectrometric methods. Immuno-analytical methods frequently suffer from low and poorly characterised detection specificity of antinitrotyrosine antibodies, while mass spectrometric methods for identification of Tyrosine nitration may be hampered by low levels of modification, and by possible changes of structure and proteolytic degradation of proteins introduced by the nitration. Moreover, no detailed, molecular characterisation of the specificity of anti-3-nitrotyrosine antibodies has been reported. In this study we describe a molecular study of the recognition specificities and affinities of two commercially available, monoclonal anti-nitrotyrosine antibodies by affinity-mass spectrometry, using different 3-nitrotyrosine containing peptides. Tyrosine-nitrated and non-nitrated substrate peptides of prostacyclin synthase (PCS), an enzyme inactivated by nitration of the active site Tyr-430 residue, were synthesised by solid-phase peptide synthesis (SPPS), purified by reversed phasehigh performance liquid chromatography (RP-HPLC) and characterised by electrospray (ESI) and matrix-assisted laser desorption-ionisation (MALDI) mass spectrometry. Binding affinities and specificities of PCS peptides with different Tyr-nitration sites and sequence mutations adjacent to Tyr-430 were determined by evaluation of anti-nitrotyrosine antibodies using an affinitymass spectrometry approach, compared to immuno-analytical determination using dot-blot and ELISA. The results showed that the antibodies may discriminate in the recognition of peptides with different N-terminal adjacent sequences to the nitrotyrosine residues, depending on the type of immunogen employed. A quantitative ELISA estimation was developed for the determination of antibody binding by Tyrosine-nitrated peptides.