Time- and pH-dependent copper binding to Aβ(1-16) peptide: An electrospray ionization-mass spectrometric approach

An elevated concentration of copper ions in the brain of Alzheimer’s disease patients has been reported in many studies and might be associated with an increased aggregation of b-amyloid (Ab) peptides. In the present work, the interaction with copper ions of a model b-amyloid peptide, Ab(1–16), was investigated by electrospray ionization-mass spectrometry (ESI–MS) at two pH values, 7.4 and 6.6, as well as at various peptide: copper ion ratios in the first minutes after components mixing and time intervals. Our results indicated that copper ions specifically bound to Ab(1–16) peptide in solution and that the complex formation increased with time. Once formed in solution, Cu2?-Ab(1–16) complexes could easily be detected in the gas phase by ESI–MS. The pH shift from 7.4 to 6.6 only slightly influenced the Cu2? binding to Ab(1–16). No oligomerization of Ab(1–16) peptide was noticed in the first minutes of copper-peptide interaction

Új tömegspektrometriás módszerek kifejlesztése és alkalmazása a peptid- és fehérjekutatásban = Development and application of mass spectrometric techniques for the analysis of peptides and proteins

Kutatásaink során peptidek, proteinek, polilizinek és molekuláris komplexek tömegspektrometriás vizsgálatával foglalkoztunk. Molekuláris komplexek körében mind szolvatált fémionokkal, mind pedig antitest-antigén komplexekkel foglalkoztunk, ezeket sikeresen előállítottunk és tömegspektrometriával jellemeztünk. Legfontosabb eredményünk egy olyan új peptidfragmentációs modell kidolgozása, mely peptidek tömegspektrumának, tandem tömegspektrumának elméleti meghatározását teszi lehetővé. A modell alapján egy olyan új szoftvert fejlesztettünk ki, mely a világon egyedülálló, prediktív jellegű és a gyakorlatban jól használható. A modell "szemi-empirikus" megoldás: az elméleti, általunk kifejlesztett MassKinetics algoritmus alapján működik, a fragmentáció leírásához szükséges fizikai, fiziko-kémiai paramétereket viszont empirikusan meghatározott paraméterekkel helyettesíti. A program a proteomika eszköztárát jelentősen továbbfejleszti, szabadalmaztatást követően világszerte elérhetővé tesszük. | We have studied peptides, proteins, polylysins and various molecular complexes by mass spectrometry. In the latter class we have produced and characterized both solvated metal ions and antibody-antigen complexes. Our most important result is development of a new peptide fragmentation model which allows theroretical prediction of tandem mass spectra. Based on this model a new software has been developed which is unique, has predictive value and can be used easily. It is a "semi-empirical" method, as it is based on the theoretical MassKinetics algorithm, but the physical and physico-chemical parameters needed to describe peptide fragmentation are determined by an empirical approach. The program can be advantageously used in the proteomics field and will be made widely available after patenting

Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate

Transglutaminase 2 (TG2) is a ubiquitously expressed multifunctional protein with Ca2+-dependent transamidase activity forming protease resistant Nε-(γ-glutamyl)lysine crosslinks between proteins. It can also function as an isopeptidase cleaving the previously formed crosslinks. The biological significance of this activity has not been revealed yet mainly because of the lack of protein based method for its characterization. Here we report development of a novel kinetic method for measuring isopeptidase activity of human TG2 by monitoring decrease in the fluorescence polarisation of a protein substrate previously formed by crosslinking fluorescently labelled glutamine donor FLpepT26 to S100A4 at a specific lysine residue. The developed method could be applied to test mutant enzymes and compounds which influence isopeptidase activity of TG2

Cell-penetrating conjugates of pentaglutamylated methotrexate as potential anticancer drugs against resistant tumor cells

The emerging resistance of tumor cells against methotrexate (MTX) is one of the major limitations of the MTX treatment of tumorous diseases. The disturbance in the polyglutamation which is a main step in the mechanism of methotrexate action is often the reason of the resistance. Delivery of polyglutamylated MTX into cells may evade the mechanisms that are responsible for drug resistance. In this study conjugates of methotrexate and its pentaglutamylated derivatives with cell-penetrating peptides – penetratin and octaarginine – were investigated. The cellular-uptake and in vitro cytostatic activity of conjugates were examined on breast cancer cell cultures (MDA-MB-231 as resistant and MCF-7 as sensitive cell culture). These cell cultures showed very different behaviour towards the conjugates. Although the presence of pentaglutamyl moiety significantly decreased the internalisation of conjugates, some of them were significantly active in vitro. All of the conjugates were able to penetrate in some extent into both cell types, but only the conjugates of penetratin showed in vitro cytostatic activity. The most effective conjugates were the MTX-Glu5-Penetratin(desMet) and MTX-Glu5-GFLG-Penetratin(desMet). The latter was effective on both cell cultures while the former was active only on the resistant tumor cells. Our results suggest that the translocation of polyglutamylated MTX may be a new way to treat sensitive and more importantly resistant tumors. While both penetratin and octaarginine peptides were successfully used to deliver several kinds of cargos earlier in our case the activity of penetratin conjugates was more pronounced

Quantitative comparison of tandem mass spectra obtained on various instruments

The similarity between two tandem mass spectra, which were measured on different instruments, was compared quantitatively using the similarity index (SI); defined as the dot product of the square root of peak intensities in the respective spectra. This function was found to be useful for comparing energy dependent tandem mass spectra obtained on various instruments. Spectral comparisons show the similarity index in a 2D “heat map”; indicating which collision energy combinations result in similar spectra; and how good this agreement is. The results and methodology can be used in the pharma industry to design experiments and equipment well suited for good reproducibility. We suggest that to get good long term reproducibility, it is best to adjust the collision energy to yield a spectrum very similar to a reference spectrum. It is likely to yield better results than using the same tuning file, which e.g. does not take into account that contamination of the ion source due to extended use may influence instrument tuning. The methodology may be used to characterize energy dependence on various instrument types; to optimize instrumentation; and to study the influence or correlation between various experimental parameters

Hatékony tumorellenes készítmények előállítása target és drug molekulák kombinációjával = Synthesis of effective antitumoricidal compounds with the combination of target and drug molecules

Az irányított tumorterápiában alkalmazható új vegyületeket terveztünk és állítottunk elő. GnRH-III dimer származékok nagyobb tumorellenes hatást és kisebb endokrin aktivitást mutattak, mint a monomer. Enzim stabilitásuk szintén fokozódott. In vivo tumorellenes hatásukat HT-29 vastagbél tumort hordozó egereken tesztelve 50%-os gátlást értünk el. GnRH-I illetve GnRH-II és GnRH-III hormone peptideket tartalmazó vegyes dimereket is előállítottunk. T47-D emlőtumor sejteken 80% fölötti gátlást tapasztaltunk egy GnRH-I és GnRH-III peptidet tartalmazó dimer esetén. A dimerek szabadalmaztatása folyamatban van. A GnRH-III hormont hatóanyagok szállítására is felhasználtuk. Antraciklineket kapcsoltunk hozzá észter-, hidrazon- oxim és amidkötéssel. Az utobbi kivételével valamennyi konjugátum jelentős in vitro tumorellenes hatást mutatott. A daunorubicin-GnRH-III oximkötésű konjugátumot választottuk ki in vivo kísérletekhez C26 egér vastagbéltumort hordozó egereken. Ha a kezelést a 4. és 7. napon végeztük a tumorbeültetést követően, 40-50%-os tumornövekedés gátlást kaptunk (15 mg/kg Dau hatóanyag tartalom mellett). Ugyanakkor a konjugátum kivédte a drog toxikus hatását, és túlélésnövekedést is eredményezett a szabad droghoz képest. Ez az első olyan eredmény, ahol oximkötéssel konjugált daunorubicin származékkal tumorellenes hatást értek el. Tuftsin-szerű hordozómolekulák alkalmazásával kemotaxison alapuló hatóanyag célbajuttatást tudtunk megvalósítani. | New derivatives for targeted cancer therapy were developed. Dimers derived from GnRH-III had higher antitumor effect on cancer cells and lower activity on LH secretion than the monomer. Enhanced enzymatic stability of the dimers was also determined. The in vivo antitumor effect of the dimers was studied on HT-29 colon tumor bearing mice and 50% tumor growth inhibition was detected. Asymmetric dimer derivatives derived from GnRH-I or GnRH-II and GnRH-III were prepared. The combination of GnRH-I and GnRH-III resulted in a superagonist compound having 80% in vitro antiproliferative effect on T47-D human breast cancer cells. GnRH-III was also used as targeting moiety for anticancer drug delivery. Anthracyclines were attached to it via ester, hydrazone, oxime or amide bond. Except amide bond containing derivatives all compounds with other type of linkage have significant antitumor activity. Oxime bond-linked Daunorubicin?GnRH-III conjugate was selected for in vivo studies on C26 murine colon carcinoma bearing mice. The results showed that the application of the conjugate prevent the toxic side effect of the free drug. The highest tumor growth inhibition (40-50%) was observed when the treatments were performed on days 4 and 7 after tumor transplantation using 15 mg Dau content in conjugate/kg body weight. This is the first study indicating significant antitumor effect of a Dau-conjugate with oxime linkage. Chemotactic drug targeting with tuftsin like carriers was also used

Célzott terápiára alkalmas új peptidkonjugátumok szintézise és alkalmazása rosszindulatú daganatok kezelésében. = Development of new peptide conjugates for targeted therapy of cancer.

Célunk az volt, hogy olyan hatóanyag szállító rendszereket állítsunk elő, amelyek a tumornövekedést gátló hatóanyagokat szelektíven juttatják a tumoros sejtekbe. Ennek érdekében előállítottunk több mint 70 vegyületet, ezen belül kb. 50 GnRH illetve oligotuftsin hordozó – hatóanyag konjugátumot. Vizsgáltuk a konjugátumok in vitro tumorellenes hatását, sejtbe jutásukat, enzim stabilitásukat, metabolizmusukat és a metabolitok DNS kötődését. Összehasonlítottuk néhány vegyület receptorkötődési képességét. Szerkezet-hatás összefüggések vizsgálatával megállapítottuk, hogy milyen szekvencia módosítások, peptid-hatóanyag közötti kötéstípusok (pl. oxim, észter, hidrazon,) eredményeznek hatékonyabb konjugátumokat. A tumorellenes hatást több hatóanyag sejtbe juttatásával is fokozni kívántuk. Ezért a konjugátumok dimerjeit, illetve több drog molekulát tartalmazó konjugátumot is előállítottunk. Vizsgáltuk a vegyületek kemotaxisát és sejtadhézióra gyakorolt hatását, amelyek fontosak a tumor metasztázisában. Az vizsgálatok megkönnyítése érdekében saját fejlesztésű kemotaxis chippet készítettünk. A tumorsejtekben kezelés hatására lejátszódó, fehérje és RNS szintű változások tanulmányozására proteomikai és RT-PCR módszereket alkalmaztunk. A kutatás adott fázisában az aktuálisan leghatékonyabb vegyületek in vivo tumorellenes hatását vizsgáltuk vastagbél tumorral beoltott egereken. Az eredményekből olyan következtetéseket vontunk le, amelyek jelentősek a további kísérletek szempontjából. | The aim of our project was the development of drug delivery systems that target anticancer drugs selectively to the tumor cells. For this purpose more than 70 new compounds, among them ca. 50 GnRH and oligotuftsin based carrier – drug conjugates, were prepared. In vitro antitumor activity, cellular uptake, enzyme stability and metabolism of the conjugates were studied. The DNA binding of the metabolites was also investigated. The receptor binding activity of several compounds was checked and compared. The structure – activity relationship studies showed the allowed substitutions in the peptide sequences. The influence of types of bond (e.g. oxime, ester, hydrazone) between the drug and carrier molecule on efficient drug release was identified. The antitumor activity of the compounds could be increased by the elevation of drug molecules (identical or different) on carrier molecules or by dimerization of the drug containing monomer conjugates. For the development of metastasis preventing compounds the chemotactic activity of the conjugates and their influence on cell adhesion were also studied. For these studies new chemotactic chips were developed. The protein or RNA based changes in the treated cells compared to untreated cells were studied by proteome analysis or by RT-PCR. The best compounds were selected for in vivo studies. The antitumor activity of the compounds was measured on colon cancer bearing mice. The results of the project are useful for the further research

Mapping the tandem mass spectrometric characteristics of citrulline containing peptides

RATIONALE Protein citrullination (deimination) is a post-translational modification of proteins converting arginine(s) to citrulline(s). “Overcitrullination” could be associated with severe pathological conditions. Mass spectrometric analysis of modified proteins is hindered by several problems. A comprehensive study of fragmentation of deiminated peptides is not yet available. In this paper we have made an attempt to describe the characteristics of these processes, based on the studies of epitope model oligopeptides derived from clinically relevant proteins. METHODS Solution of purified model peptides containing either one or two citrulline residues as well as their native variants were injected directly to the electrospray source of a high accuracy and resolution quadrupole-time of flight instrument and were analysed by tandem mass spectrometry using low-energy collision induced dissociation. RESULTS Loss of isocyanic acid from citrulline residues is a preferred fragmentation route for deiminated peptides, which yields ornithine residues in the sequence. However, simultaneous detection of both the isocyanic acid loss and sequence fragments is often compromised. A preferential cleavage site was observed between citrulline and any other following amino acids yielding intensive complementary b and y type ions. Also, citrulline positioned at the C-termini displays a preferential cleavage N-terminal to this residue yielding characteristic y1 ions. These phenomena are described here for the first time and are referred to as the “citrulline effect”. CONCLUSIONS We found that the citrulline effect is very pronounced and could be used as a complementary tool for the confirmation of modification sites in addition to losses of isocyanic acids from the protonated molecules or from fragment ions. Low collision energy applied to peptide ions having partially mobile protons reveal the site of modification by generating specific and intensive fragments of the sequence. On the other hand, fragmenting parent ions with mobile protons usually allow full sequence coverage, although citrulline-specific fragments may exhibit lower intensities compared to other fragments

Metastasis-associated S100A4 is a specific amine donor and an activity-independent binding partner of transglutaminase-2.

Transglutaminase-2 (TG2) is best known as a Ca(2+)-dependent cross-linking enzyme; however, some of its extracellular matrix-related functions are independent of its catalytic activity and include matrix remodelling, adhesion and migration. S100A4 belongs to the Ca(2+)-binding EF-hand S100 protein family and acts both intra- and extra-cellularly through binding to various partners. It regulates cell migration and its overexpression is strongly associated with metastasis and poor survival in various cancers. It has recently been suggested that TG2 mediates S100A4-dependent tumour cell migration. In the present study we provide evidence that S100A4 is an interacting partner and also a specific amine donor of TG2. TG2 incorporates a glutamine donor peptide to Lys(100) in the C-terminal random coil region of S100A4. Importantly, the enzyme activity is not necessary for the interaction: S100A4 also binds to TG2 in the presence of a specific inhibitor that keeps the enzyme in an open conformation, or to an enzymatically inactive mutant. We also found that S100A4 considerably enhances TG2-mediated adhesion of A431 epithelial carcinoma cells to the extracellular matrix. This role is independent of enzyme activity and requires the open conformation of TG2. We propose that S100A4 stabilizes the open conformation of TG2, which binds to its cell-surface receptor in this state and increases cell adhesion