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Non-proteinogenic amino acids are widely explored group of compounds due to their chemical properties and great potential of application in the combinatorial chemistry, medicinal investigation etc. Therefore the synthetic methods of their incorporation to the peptide chain are required. l-Mimosine, (S)-α-amino-β-(3-hydoxy-4-oxo-1,4-dihydropyridin-1-yl)-propanoic acid), is a plant amino acid, known to induce apoptosis in human pancreatic cancer xenografts. Here we present our investigations on the synthesis of mimosine-containing peptide and their ESI-MS/MS analysis. We successfully applied Fmoc-protected mimosine a with a free hydroxy ketone group for efficient peptide synthesis in the presence of HATU as a coupling reagent without the formation of side products. Additionally the tandem mass spectrometry analysis revealed the characteristic loss of the heterocyclic ring from mimosine residue side chain. The described method allows insertion of mimosine residue at any endo-position within a peptide sequence. The obtained results may be useful in the synthesis and mass spectrometry analysis of various mimosine-containing peptides.

In veterinary medicine, sensitive and specific markers of the early stages of renal failure still remain to be established. Podocytes could be a promising diagnostic tool in veterinary nephrology, especially in the differentiation of active pathological disease and glomerulopathies. Podocin is one of the robust proteins exploitable in detection of podocyturia. This article presents podocyte detection in urine for diagnostic purposes in veterinary medicine using a variety of methods. We describe the advantages and disadvantages of the immunohistochemical technique currently used, and of scanning microscopy, chromatography, and immunostaining. The identification of podocin-positive cells is a promising diagnostic tool in the detection of the early stages of glomerular basement membrane damage. The detection of renal failure prior to the occurrence of azotaemia is of high clinical importance from the clinical and scientific points of view.

Early detection of any preeclampsia biomarkers may lower the risk of mortality, both for a mother and a child. Our study focuses on techniques for preeclampsia biomarker identification by comparing the results of a method using liquid chromatography mass spectrometry in multiple reaction monitoring mode (LC-MS/MS) with those by the enzyme-linked immunosorbent assay (ELISA) test, as well as by comparing the obtained results with clinical data. In the proposed LC-MS/MS method a tryptic digest peptide charge derivatization strategy was used as a tool for sensitive detection of podocin, i.e., a previously discovered preeclampsia biomarker present in urine samples from pregnant women. Urine samples from pregnant women with diagnosed preeclampsia were collected at different stages of pregnancy and from healthy subjects, and then were analyzed by ELISA test and the proposed method with LC-MS/MS. Charge derivatization of the ε amino group of C-terminal lysine residues in tryptic digests by 2,4,6-triphenylpyrylium salt was performed to increase the ionization efficiency in the LC-MS/MS mode. Podocin was identified at the early stage of pregnancy, while its detection using an ELISA test was not possible. The protocol for urine sample preparation was optimized. Our results show that the proposed method by LC-MS/MS in combination with peptide charge derivatization, provides an ultrasensitive tool for diagnosis of preeclampsia, and provides earlier detection than a clinical diagnosis or ELISA test. The proposed solution may revolutionize medical diagnostics.

SALEN- and SALAN-based complexes with catalytically active metal centers are very promising small molecules to be utilized as part of antioxidant therapies. Here we discuss a modified SALAN-type molecule armed with two phosphonate groups that significantly increase its water solubility and aid to furnish mono- or dinuclear complexes with Cu2+ ions. The regulation of the SOD-mimicking (i.e., catalytic) disproportionation reaction of the superoxide radical anion (O2•−) at pH ~7.5 could be achieved by adjusting the metal-to-ligand stoichiometry as confirmed by McCord-Fridovich and pulse radiolysis tests. The higher antioxidant activity of the dicopper complex can be explained by the better access of O2•− to the copper centers and their more positive Cu(II)/Cu(I) redox potential. Simultaneously the analysis of in vitro effect on cells morphology indicates that cytotoxicity is also affected by the metal-to-ligand ratio, however, the active complex molecules do not show notable cytotoxicity that, together with the observed SOD-like activities, makes them potential candidates for antioxidant therapies.

A multiply branched peptide, H-Gly-Dap(H-Gly)-Dap(H-Gly-Dap(H-Gly))-Gly-NH2 (Dap = L-2,3-diaminopropionic acid) has been designed to furnish multi-Cu2+ centers exhibiting electrocatalytic activity in water oxidation. Each branching moiety serves as binding pocket for one cupric ion. UV–vis, CD and EPR spectroscopy suggest {NH2,N−,N−,NH2}eq set for the first two coordinated Cu2+ ions, whereas a different mode for the third that in turn changes the electrochemical behavior of the complex. This third Cu2+ triggers water oxidation at lower (~60 mV) overpotential, increased catalytic current and 4 times as high O2 evolution rate compared to the 2:1 metal-to-ligand stoichiometry.

Enzymatic 18O exchange, the well-established approach in comparative proteomics, has some disadvantages such as back exchange of labeled oxygen and overlapping the peak of a labeled peptide with isotopic peaks of an unlabeled one. Herein we demonstrated a simple procedure in which samples digested with a trypsin (with and without H218O) were reacted with unlabeled and quadrupled 13C-labeled pyrylium salt respectively which results in formation of pyridinium cations. Thus, each isobarically labeled peptide containing zero or four 13C atoms in the mass reporter group, during tandem MS/MS forms an unique reporter ion useful for a relative quantitation. Such a sample treatment improves the signal to noise ratio, reduces overlapping of the isotopic peaks and completely eliminates the back exchange problem.

Mass spectrometry methods are commonly used in the identification of peptides and biomarkers. Due to a relatively low abundance of proteins in biological samples, there is a need for the development of novel derivatization methods that would improve MS detection limits. Hence, novel fluorescent N–hydroxysuccinimide esters of dihydro-[1,2,4]triazolo[4,3-a]pyridin-2-ium carboxylates (Safirinium P dyes) have been synthesized. The obtained compounds, which incorporate quaternary ammonium salt moieties, easily react with aliphatic amine groups of peptides, both in solution and on the solid support; thus, they can be applied for derivatization as ionization enhancers. Safirinium tagging experiments with ubiquitin hydrolysate revealed that the sequence coverage level was high (ca. 80%), and intensities of signals were enhanced up to 8-fold, which proves the applicability of the proposed tags in the bottom–up approach. The obtained results confirmed that the novel compounds enable the detection of trace amounts of peptides, and fixed positive charge within the tags results in high ionization efficiency. Moreover, Safirinium NHS esters have been utilized as imaging agents for fluorescent labeling and the microscopic visualization of living cells such as E. coli Top10 bacterial strain.

Amadori products (deoxyfructosyllysine derivatives) that can selectively interact with phenylboronic acids and borate ions were synthesized. The intramolecular interactions between the fructosyl moiety and phenylboronic acid incorporated into various positions of the peptide chain were investigated using high-resolution mass spectrometry (HR–MS), circular dichroism (CD), and nuclear magnetic resonance (NMR).

Detection of podocytes in urine might serve as a useful diagnostic tool in both primary and secondary glomerular diseases. The utility of podocyturia has been confirmed for both pre-eclampsia and glomerulonephritis. Here, we present a new and sensitive method for qualitative LC-MS-multiple-reaction-monitoring (MRM) analysis of podocin, serving as a podocyturia biomarker in urine sediments. The following podocin tryptic peptides with the 169LQTLEIPFHEIVTK182, 213AVQFLVQTTMK223, 240SIAQDAK246, and 292MIAAEAEK299 sequences were applied as a model. The selective chemical derivatization of the &epsilon, amino group of C-terminal lysine residue in tryptic peptides, by 2,4,6-triphenylpyrylium salt (TPP) as a fixed charge tag, was employed to increase the ionization efficiency, in routine ESI-MS analysis. Additionally, the generation of a reporter ion, in the form of a protonated 2,4,6-triphenylpyridinium cation, makes the derivatized peptide analysis in the MRM mode unambiguous. Identification of derivatized and non-derivatized peptides were performed, and the obtained results suggest that the peptide with the 292MIAAEAEK299 sequence may serve as a marker of podocyturia.

Kidneys play a crucial role in maintaining metabolic homeostasis in a body. Serum creatinine concentration is a simple test used as an indicator of renal function. One of the known ways of quantifying creatinine concentration is the liquid chromatography-mass spectrometry (LC-MS) method, using an isotopically labeled analog of creatinine as an internal standard. Unfortunately, such isotope-labeled analogs are expensive and their synthesis is complex. Here we demonstrate a facile preparation of deuterated analogues of creatinine, via the H/D exchange of hydrogens located at the &alpha, carbon (&alpha, C) of the N-methylated amino acid part, under basic conditions. The stability of retrieved isotopologues was analyzed under both neutral or acidic conditions, and the results revealed that the introduced deuterons do not undergo back-exchange. In addition, the coelution of deuterated and non-deuterated forms under acidic and neutral conditions was observed. The prepared isotopologues were successfully applied in the quantitative LC-MS analysis of urine samples, and the results demonstrated that the presented strategy is novel and inexpensive, and that the quantification correlates with the commonly used Jaffe test method.

High complexity of cell and tissue proteomes limits the investigation of proteomic biomarkers. Therefore, the methods of enrichment of some chemical groups of peptides including thiopeptides are important tools that may facilitate the proteomic analysis by reducing sample complexity and increasing proteome coverage. Here, we present a new method of cysteine-containing tryptic peptide enrichment using commercially available TentaGel R RAM resin modified by the linker containing the maleimide group, allowing thiol conjugation. The captured tryptic peptides containing lysine residue were then tagged by 2,4,6-triphenylpyrylium salt to form 2,4,6-triphenylpyridinium derivatives, which increases the ionization efficiency during mass spectrometry analysis. This makes it possible to conduct an ultrasensitive analysis of the trace amount of compounds. The proposed strategy was successfully applied in the enrichment of model tryptic podocin peptide and podocin tryptic digest.

Mimosine is a non-protein amino acid with various properties, such as antibacterial, anti-inflammatory, anti-cancer and anti-virus among others. Due to its structural similarity with deferiprone (DFP), mimosine is a potential excellent metal chelator. In the present work, we combine experimental and theoretical (DFT) approaches in order to investigate the properties of mimosine peptides. Six different peptides were synthesized and their complex stoichiometry and stability were characterized by means of UV-Vis spectrophotometry. Then, the binding mode and self-assembly features of the peptides were evaluated using a DFT approach, taking into account different number of mimosine amino acids and varying the length of the spacer between the mimosine residues, and there was good agreement between experimental data and computational calculations. Further elucidations of the structural properties of these peptides allowed us to propose improvements in the structure of the mimosine moiety which can lead to enhanced affinity for high-valent metals. Moreover, we demonstrate that these peptides show an anti-microbial activity against Gram positive bacteria that is enhanced by the formation of a complex with iron(iii) ions. The mimosine peptides could be an alternative to antimicrobial peptides (AMPs), which are expensive and susceptible to proteolytic degradation. In summary, in the present work, we propose a new generation of multipurpose mimosine-based peptides as new metal self-assembly chelators which could be a turning point in biomedical and nanotechnological applications.

We report herein a novel ChemMatrix&reg, Rink resin functionalised with two phenylboronate (PhB) moieties linked on the N-&alpha, and N-&epsilon, amino functions of a lysine residue to specifically capture deoxyfructosylated peptides, compared to differently glycosylated peptides in complex mixtures. The new PhB-Lys(PhB)-ChemMatrix&reg, Rink resin allows for exploitation of the previously demonstrated ability of cis diols to form phenylboronic esters. The optimised capturing and cleavage procedure from the novel functionalised resin showed that only the peptides containing deoxyfructosyl-lysine moieties can be efficiently and specifically detected by HR-MS and MS/MS experiments. We also investigated the high-selective affinity to deoxyfructosylated peptides in an ad hoc mixture containing unique synthetic non-modified peptides and in the hydrolysates of human and bovine serum albumin as complex peptide mixtures. We demonstrated that the deoxyfructopyranosyl moiety on lysine residues is crucial in the capturing reaction. Therefore, the novel specifically-designed PhB-Lys(PhB)-ChemMatrix&reg, Rink resin, which has the highest affinity to deoxyfructosylated peptides, is a candidate to quantitatively separate early glycation peptides from complex mixtures to investigate their role in diabetes complications in the clinics.

In earlier works we have described that mice immunized with outer membrane protein OmpC survive the challenge with live Shigella flexnerii 3a. We have also identified conformational epitope of this protein, that was recognized by mice antibodies. The aim of current work was to investigate whether synthetic OmpC epitope homologs can elicit immunological response sufficient in protecting mice against shigellosis. Several linear peptides containing RYDERY motif were synthesized and conjugated to poly-lysine. These conjugates appeared to be poor immunogens and to boost the immunological response an addition of the adjuvant (MPL) was required. Unfortunately, the MPL alone caused a very high immunological reaction that was masking response to peptidic epitope. Under those circumstances we used tetanus toxoid (TT) as the carrier protein for the peptides and the agent stimulating immunological response. Series of cyclic peptides, homologs of the OmpC main epitope were synthesized and conjugated to TT. The loop size in cyclic peptides varied by number of glycine residues, i.e., 1-3 residues added to the GLNRYDERYIGK motif. The linear GLNRYDERYIGC-TT was also prepared as the control. The latter conjugate gave the highest immunological response, followed by the cyclic-GGLNRYDERYIGC-TT and cyclic-GLNRYDERYIGC-TT. The third peptide, cyclic-GGGLNRYDERYIGC-TT, gave a very low response, although it was the most resistant to proteolysis. However, antibodies obtained against cyclic-GGLNRYDERYIGC-TT were more potent to recognize both OmpC and Shigella flexnerii 3a cells than the antibodies against linear GLNRYDERYIGC-TT. Furthermore, the monoclonal antibodies raised against linear GLNRYDERYIGC-TT showed 20-fold lower dissociation constant (KD) than the naturally occurring polyclonal antibodies from umbilical cord sera. Monoclonal antibodies also gave a weaker signal in electron microscope than mice and human polyclonal antibodies. In overall, our results point to cyclic peptides as better candidates for a vaccine development, since they are eliciting production of the higher affinity antibodies against Shigella cells and OmpC.

Relations between structural modifiactions and SOD-like activity of four branched CuII-metallopeptides based on l-2,3-diaminopropionic acid are reported. The IC50 values (McCord–Fridovich method) illustrate that enhanced SOD-like activity is warranted by exclusive N-terminal His localization that surmounts the driving force by the CuII/I formal potential.

Recently, we developed a novel non-fragmenting quaternary ammonium ionization tag for the mass spectrometric sensitive sequencing of peptides, based on the N-spiro proline residue (5-azoniaspiro[4.4.]nonyl-carbonyl). Herein, we present an unexpected racemization and the hydrogen-deuterium exchange (HDX) at the α-C atom of the proline derivative under basic aqueous conditions (1% water solution of triethylamine). The deuterium atom, substituted for the α-C atom, does not undergo back-exchange under acidic aqueous conditions which makes the deuterated isotopologue a promising stabile isotope-coded internal standard for quantitative analysis by mass spectrometry. The applicability of the prepared isotopologues of the quaternary ammonium salt labeled peptides for quantification experiments using the isotopic dilution method was also examined.

Isobaric labeling quantification of peptides has become a method of choice for mass spectrometry-based proteomics studies. However, despite of wide variety of commercially available isobaric tags, none of the currently available methods offers significant improvement of sensitivity of detection during MS experiment. Recently, many strategies were applied to increase the ionization efficiency of peptides involving chemical modifications introducing quaternary ammonium fixed charge. Here, we present a novel quaternary ammonium-based isobaric tag for relative and absolute quantification of peptides (QAS-iTRAQ 2-plex). Upon collisional activation, the new stable benzylic-type cationic reporter ion is liberated from the tag. Deuterium atoms were used to offset the differential masses of a reporter group. We tested the applicability of QAS-iTRAQ 2-plex reagent on a series of model peptides as well as bovine serum albumin tryptic digest. Obtained results suggest usefulness of this isobaric ionization tag for relative and absolute quantification of peptides.

Quaternary ammonium salts (QAS), both linear and bicyclic, are often utilized to improve the mass spectrometry (MS) analysis of peptides by fixing a permanent positive charge on the analyzed molecule. However, during collision-induced dissociation (CID) experiments, QAS undergo unwanted side reactions—Hofmann elimination as well as a tertiary amine loss— rendering the data interpretation complicated. In this work, we present 2-thia- and 2-oxa-5-azoniaspiro[4.4]nonyl groups as heterocyclic derivatives of the highly stable ionization group, 5-azoniaspiro[4.4]nonyl, for a sensitive peptide analysis by MS. Due to the permanent positive charge, labeled peptides are characterized by enhanced ionization efficiency during electrospray mass spectrometry (ESI-MS) conditions. Moreover, interpretation of the CID fragmentation of labeled peptides is facilitated since a series of generated fragmentation ions enable a complete sequence coverage. Introduction of a heteroatom into the 5-azoniaspiro[4.4]nonyl scaffold allows for liberation of a stable reporter ion which could be used in selected reaction monitoring (SRM)-targeted quantification experiments. Additionally, we synthesized a deuterated analog of the tag for LC-SRM-targeted quantitative analysis. The obtained results indicate the general usefulness of the proposed heterocyclic quaternary ammonium ionization tag for sequencing and quantification of peptides. Graphical abstractNew reagents based on the structure of the 5-azoniaspiro[4.4]nonyl tag for peptide analysis by tandem mass spectrometry Electronic supplementary material The online version of this article (10.1007/s00216-017-0771-2) contains supplementary material, which is available to authorized users.

Peptide modification by a quaternary ammonium group containing a permanent positive charge is a promising method of increasing the ionization efficiency of the analyzed compounds, making ultra-sensitive detection even at the attomolar level possible. Charge-derivatized peptides may undergo both charge remote (ChR) and charge-directed (ChD) fragmentation. A series of model peptide conjugates derivatized with N,N,N-triethyloammonium (TEA), 1-azoniabicyclo[2.2.2]octane (ABCO), 2,4,6-triphenylopyridinium (TPP) and tris(2,4,6-trimetoxyphenylo)phosphonium (TMPP) groups were analyzed by their fragmentation pathways both in collision-induced dissociation (CID) and electron-capture dissociation (ECD) mode. The effect of the fixed-charge tag type and peptide sequence on the fragmentation pathways was investigated. We found that the aspartic acid effect plays a crucial role in the CID fragmentation of TPP and TEA peptide conjugates whereas it was not resolved for the peptides derivatized with the phosphonium group. ECD spectra are mostly dominated by cn ions. ECD fragmentation of TMPP-modified peptides results in the formation of intense fragments derived from this fixed-charge tag, which may serve as reporter ion.

We conducted a thorough study of Cu2 + complex formation equilibria with Irbesartan and Losartan, the two primary drugs for the cure of cardiovascular diseases, with the aim of recognising if these drugs could exert a chelating action towards Cu2 +. We used different complementary techniques to gain a clear picture of the involved protonation and complexation equilibria. The low solubility in water of the ligands and of the formed metal complexes prevented the use of water as solvent, so we had to perform the measurements in mixed methanol-water solvents. Further, we studied the related equilibria with Zn2 + for evaluating a potential interference of this essential metal ion, largely present in biological fluids. Our study provided a strong evaluation of the formed complexes and of the relative stability constants. The binding of both metal ions takes place through the tetrazole moiety except for the Zn2 +-Irbesartan system. In this last case, NMR measurements gave evidence of a tautomeric equilibrium involving the imidazole ring and the aliphatic chain. The estimated complexation model, and the related stability constants, allowed a speciation study in human plasma, based on a number of simplifying assumptions, which remarked that both drugs, Losartan and Irbesartan, could exert a chelating action, scavenging non-negligible amounts of Cu2 + from the organism.

The imidazole ring of histidine (His) and the methylcarboxylate function of aspartic acid (Asp) are crucial for Cu coordination in enzymes, artificial proteins and other bioinspired systems. In order to elucidate the effect of Asp at the C-terminus, a new branched peptide, H-Gly-Dap(H-Gly)-Asp-NH2 (2GD, Dap = 2,3-diaminopropionic acid) has been synthesized to ligate CuII. While at pH < 8, 2GD favours dimeric complexes at 1 : 1 metal-to-ligand ratio, at increasingly alkaline pH the Asp at the C-terminus creates a {NH2,N−,N−,O−}eq set and the dominance of mononuclear CuLH−2 and CuLH−3 species, according to potentiometry, molecular spectroscopy (UV-visible, circular dichroism and X-band electron paramagnetic resonance) and ESI-MS analysis. The changes in speciation with pH are accompanied by shifts in the CuIII/II redox current peaks that were analyzed by square wave voltammetry (SWV) and allowed the calculation of the pKa values for the CuII and CuIII forms. The 2GD complex exhibits electrocatalytic activity in water oxidation in phosphate buffer. However, when Asp residues are present at all three termini to produce 3D (H-Asp-Dap(H-Asp)-Asp-NH2), the electrocatalysis of water oxidation decreases and, if present only at the N-termini in 2DG (H-Asp-Dap(H-Asp)-Gly-NH2), it ceases. As for CuII ligated by 2GD, a TOF of ∼16 s−1 was calculated at pH = 11 in 0.2 M phosphate electrolyte, which is crucial for catalysis, but also acts as inhibitor anion according to cyclic voltammetry observations. The system is highly tolerant to the presence of chloride, which is a feature of practical importance in efficient water oxidation catalysis.

Dentin matrix protein 1 (DMP1) is an acidic, extracellular matrix protein essential for biomineralization of calcium phosphate, in bone and dentin. It is proteolytically processed into two fragments, 44K and 56K. Recently, the presence of DMP1 was noticed in inner ear, specifically in otoconia, which are calcium carbonate biominerals involved in sensing of balance. In this study, the solution structure and biomineralization activity of otoconial 44K and 56K fragments toward calcium carbonate were investigated. The results of analytical ultracentrifugation, circular dichroism, and gel filtration indicated that DMP1 fragments are disordered in solution. Notably, 56K formed oligomers in the presence of calcium ions. It was also observed that both fragments influenced the crystal growth by in vitro biomineralization assay and scanning electron microscopy. In addition, they sequester the calcium ions during the calcite formation. Calcium carbonate crystals precipitated in vitro changed their size and shape in the presence of DMP1 fragments. Oligomerization propensity of 56K may significantly enhance this function. Our study indicates that intrinsically disordered DMP1 has a previously unknown regulatory function for biomineralization of otoconia.

Mass spectrometry methods are commonly used in the identification of peptides and biomarkers. Due to a relatively low abundance of proteins in biological samples, there is a need for the development of novel derivatization methods that would improve MS detection limits. Hence, novel fluorescent N&ndash, hydroxysuccinimide esters of dihydro-[1,2,4]triazolo[4,3-a]pyridin-2-ium carboxylates (Safirinium P dyes) have been synthesized. The obtained compounds, which incorporate quaternary ammonium salt moieties, easily react with aliphatic amine groups of peptides, both in solution and on the solid support, thus, they can be applied for derivatization as ionization enhancers. Safirinium tagging experiments with ubiquitin hydrolysate revealed that the sequence coverage level was high (ca. 80%), and intensities of signals were enhanced up to 8-fold, which proves the applicability of the proposed tags in the bottom&ndash, up approach. The obtained results confirmed that the novel compounds enable the detection of trace amounts of peptides, and fixed positive charge within the tags results in high ionization efficiency. Moreover, Safirinium NHS esters have been utilized as imaging agents for fluorescent labeling and the microscopic visualization of living cells such as E. coli Top10 bacterial strain.

The nonapeptide fragment of the HLA-DR molecule, located in the exposed loop of the alpha-chain (164-172), having the VPRSGEVYT sequence, suppresses the immune response. Based on the three-dimensional structure of the HLA-DR superdimer, we designed a new cyclodimeric analog in which the two parallel peptide chains of VPRSGEVYT sequence are linked through their C-termini by spacer of (Gly5 )2 -Lys-NH2 and the N-termini are also linked by poly(ethylene glycol). The (VPRSGEVYTG5 )2 K-resin analog was synthesized using solid-phase peptide synthesis protocols. The cyclization was achieved by cross-linking the N-terminal positions of the dimeric peptide, attached to a MBHA resin, with alpha, omega-bis (acetic acid) poly(ethylene glycol), activated by esterification with pentafluorophenol. Our results demonstrate that the cyclodimerization of VPRSGEVYT results in enhanced immunosuppressive activity of the peptide. Mass spectrometry fragmentation analysis of the obtained cyclodimeric peptide is also presented. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

The synthesis of 5-hydroxy-2-(hydroxymethyl)pyridin-4(1H)-one (P1) is presented, together with the evaluation of its coordination ability towards Fe(3+), studied by a combination of chemical, computational, and animal approaches. The use of complementary analytical techniques has allowed us to give evidence of the tautomeric changes of P1 as a function of pH, and to determine their influence on the coordinating ability of P1 towards Fe(3+). The pFe(3+) value 22.0 of P1-iron complexes is noticeably higher than that of deferiprone (20.6), one of the three clinical chelating agents in therapeutic use for iron overload diseases. This is due on one side to the tautomeric change to the catechol form, and on the other to the lower protonation constant of the OH group. Bio-distribution studies on mice allowed us to confirm in vivo the efficacy of P1. Furthermore the coordinating ability toward Al(3+), Cu(2+) and Zn(2+) has been studied to evaluate the possible use of P1 against a second toxic metal ion (Al(3+)), and to envisage its potential influence on the homeostatic equilibria of essential metal ions. The chelating ability of P1 toward these ions, not higher than that of the corresponding deferiprone, contributes to render P1 a more selective iron chelator.

No sensitive method for diagnosing early kidney dysfunction in horses has been identified so far. Many studies carried out in humans and small animals show that podocin can be useful to diagnose various kidney diseases, mainly affecting the glomeruli. The aim of this study was to perform a qualitative and quantitative analysis of podocin in urine samples obtained from healthy horses, horses with clinical kidney dysfunction and horses at risk of acute kidney injury. The study objectives aimed to assess: (1) whether the selected podocin tryptic peptide for LC-MS-MRM allows for podocin detection in horse; and (2) whether the species-specific ELISA test makes this detection possible as well;, (3) whether the chosen methods are sensitive enough to detect kidney dysfunction and glomerular injury, (4) whether the results of the tests applying both methods correspond with one another, (5) whether the results correlate with the hematological and biochemical data. The signals that may indicate the presence of trypsin fragments of podocin were found in three healthy horses, all the horses diagnosed with kidney dysfunction and half of the animals at risk for acute kidney injury. The concentration of podocin, diagnosed with the ELISA test was as follows: from 0.19 to 1.2 ng/ml in healthy animals, from 0.19 to 20.0 ng/ml in AKI horses, from 0.29 to 5.71 ng/ml in horses at risk for acute kidney injury. The results of both methods corresponded significantly. Podocin may be a potential biomarker of clinical kidney disease in horses and may be used in the detection of glomerular injury. However, its use is limited by the possibility of physiological podocyturia. LC-MS-MRM seems to be a more sensitive method to evaluate the presence of podocin than the ELISA test, whilst selected tryptic peptides of podocin appear to apply to horses. The ELISA test showed greater effectiveness in excluding the disease than in confirming it.

Proteinogenic amino acids were derivatized via coupling reactions with N-hydroxysuccinimide esters of Safirinium dyes. The obtained compounds were fully characterized by 1H and 13C NMR, IR, and ESI-MS spectral techniques. Elemental analysis, as well as the crystal structure analysis of exemplary compounds, were performed. The absorption and fluorescent properties of the obtained derivatives have been systematically investigated. The optical properties and very good water solubility allowed for the development of a simple method for the determination of exemplary amino acids by means of micellar electrokinetic chromatography. Moreover, a new class of fluorescent peptide building blocks comprising a spiro-conjugated Safirinium-proline system was obtained. Due to the presence of a permanent positive charge, Safirinium-amino acid dervatives are potential candidates for signal enhancers in MS proteomic analysis. The applicability of the examined ionization tags in signal enhancement and peptide sequencing was proved. The obtained results provide an experimental basis for the development of a new approach for trace amino acids, peptides, and proteins detection by means of absorption, fluorescence, and mass spectrometry.

The dominant vector of dengue and Zika diseases is a female Aedes aegypti mosquito. Its reproduction is controlled by the formation of an active heterodimer complex of the 20-hydroxyecdysone receptor (EcR) and Ultraspiracle protein (Usp). Although EcR exhibits a structural and functional organization typical of nuclear receptors (NRs), the EcR C-terminus has an additional F domain (AaFEcR) that is rarely present in the NRs superfamily. The presence of F domains is evolutionarily not well conserved in the NRs. The structure-function relationship of EcR F domains in arthropods is unclear and enigmatic. To date, there have been no data concerning the structure and function of AaFEcR. Our results showed that AaFEcR belongs to a family of intrinsically disordered proteins (IDPs) and possesses putative pre-molten globule (PMG) characteristics. Unexpectedly, additional amino acid composition in silico analyses revealed the presence of short unique repeated Pro-His clusters forming an HGPHPHPHG motif, which is similar to those responsible for Zn2+ and Cu2+ binding in histidine-proline-rich glycoproteins (HPRGs). Using SEC, SV-AUC and ESI-TOF MS, we showed that the intrinsically disordered AaFEcR is able to bind metal ions and form complexes with these ions. Our studies provide new insight into the structural organization and activities of the F domains of NRs. This unique for the F domains of NRs ion-binding propensity demonstrated by the AaFEcR domain may be a part of the ecdysteroid receptor's mechanism for regulating the expression of genes encoding oxidative stress-protecting proteins.

Enzymatic

This work reports the synthesis, characterization and study of the complex formation equilibria of four new bis-kojic acid chelators with Fe3+, Al3+, Cu2+ and Zn2+. Based on previous encouraging results with tetradentate kojic acid derivatives, these ligands were designed with the aim of evaluating how acidic groups in the linker can affect both protonation constants and Fe3+ coordinating properties. Fe3+ and Al3+ complexation gave evidence of high metal-sequestering capacity, above all with the first metal ion. Complex formation equilibria with the essential metal ions Cu2+ and Zn2+ were furthermore studied to evaluate any disturbance of these chelating agents on the homeostatic equilibria of the essential metal ions. A multiplicity of techniques – potentiometry, UV-visible spectrophotometry, NMR spectroscopy and ESI-MS (electrospray ionization-mass spectrometry) – have enabled the characterization of the ligands, their corresponding metal complexes, together with an exhaustive analysis of the protonation and complex equilibria.

The bicyclic amines in the form of cryptands, the crown ether analogs, were used in the synthesis of cryptando-peptidic conjugates with simultaneous formation of quaternary ammonium nitrogen moiety. A series of model cryptando-peptidic conjugates at the peptide N-terminus was efficiently prepared by the standard Fmoc solid phase synthesis. Tandem mass spectrometric analysis of the obtained conjugates has shown the specific fragmentation pattern during MS/MS experiment. The obtained cryptandic quaternary ammonium group undergoes the Hofmann elimination during collision-induced dissociation fragmentation followed by the ethoxyl group elimination. The presented quaternization of cryptands by iodoacetylated peptides is relatively easy and compatible with standard solid-phase peptide synthesis. Additionally, the applicability of such peptide derivatives and their isotopologues selectively deuterated at the α-carbon in the quantitative LC-MS analysis was analyzed.

N-substituted glycines constitute mimics of natural amino acids that are of great interest in the peptide-based drug development. Peptoids-oligo(N-substituted glycines) have been recently demonstrated to be highly active peptidomimetics in biological systems, resistant to proteolytic degradation. We developed a method of the deuterium labeling of peptidomimetics containing N-substituted glycine residues via H/D exchange of their α-carbon hydrogen atoms. The labeling was shown to be easy, inexpensive, and without the use of derivatization reagents or the need for a further purification. The deuterons introduced at the α-carbon atoms do not undergo a back exchange under acidic conditions during liquid chromatography mass spectrometry (LC-MS) analysis. The LC-MS analysis of a mixture of isotopologues revealed a co-elution of deuterated and nondeuterated forms of the peptidomimetics, which may be useful in the quantitative isotope dilution analysis of peptoids and other derivatives of N-substituted glycines.

Quantitative analysis by liquid chromatography-mass spectrometry (LC-MS) is frequently based on the application of isotopically labeled standards which usually have to be specially synthesized. Although the synthesis of deuterated isotopologues is relatively inexpensive, they are not considered as good internal standards due to the possible deuterium effect on the retention time during LC-MS analysis. We developed a method of deuterium labeling of denatonium benzoate (Bitrex) via H/D exchange of its α-carbon hydrogen atoms in CH2 group situated between carbonyl and quaternary ammonium groups. The proposed strategy is rapid, cost-efficient, and does not require derivatization reagents or further purification. The LC-MS analysis of isotopologues revealed that the introduced deuterons do not undergo back exchange under acidic conditions, and the co-elution of deuterated and non-deuterated forms is observed. The obtained deuterated standard was applied in the quantitative LC-MS analysis of Bitrex in commercially available household products. Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8815-y) contains supplementary material, which is available to authorized users.

Recently, we described a process of trypsin-assisted peptide splicing of analogs of trypsin inhibitor SFTI-1, that seems to be very similar to proteasome-catalyzed peptide splicing. Here, we show, for the first time, that a peptide-peptoid hybrid (peptomer) can also be spliced by trypsin. Incubation of a double sequence SFTI-1 analog, containing two peptoid monomers, with equimolar amount of trypsin leads to formation of monocyclic peptomer as the main product. We proved that the peptide bond formed by a peptoid monomer is not only digested by trypsin but also participates in the enzyme-assisted splicing process.