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Aldoses and ketoses can glycate proteins yielding isomeric Amadori and Heyns products, respectively. Evidently, d-fructose is more involved in glycoxidation than d-glucose favoring the formation of advanced glycation endproducts (AGEs). While Amadori products and glucation have been studied extensively, the in vivo effects of fructation are largely unknown. The characterization of isomeric Amadori and Heyns peptides requires sufficient quantities of pure peptides. Thus, the glycated building block Nα-Fmoc-Lys[Nε-(2-deoxy-d-glucos-2-yl),Nε-Boc]-OH (Fmoc-Lys(Glc,Boc)-OH), which was synthesized in two steps starting from unprotected d-fructose and Fmoc-l-lysine hydrochloride, was site-specifically incorporated during solid-phase peptide synthesis. The building block allowed the synthesis of a peptide identified in tryptic digests of human serum albumin containing the reported glycation site at Lys233. The structure of the glycated amino acid derivatives and the peptide was confirmed by mass spectrometry and NMR spectroscopy. Importantly, the unprotected sugar moiety showed neither notable epimerization nor undesired side reactions during peptide elongation, allowing the incorporation of epimerically pure glucosyllysine. Upon acidic treatment, the building block as well as the resin-bound peptide formed one major byproduct due to incomplete Boc-deprotection, which was well separated by reversed-phase chromatography. Expectedly, the tandem mass spectra of the fructated amino acid and peptide were dominated by signals indicating neutral losses of 18, 36, 54, 84 and 96 m/z-units generating pyrylium and furylium ions.

α-Aminoxy peptides represent an interesting group of peptidomimetics with high proteolytic stability and the ability to fold into specific, predictable secondary structures. Here, we present a series of hybrid peptides consisting of α-aminoxy acids and α-amino acids with cationic and aromatic, hydrophobic side chains in an alternating manner synthesized using an efficient protocol that combines solution- and solid-phase synthesis. 2D ROESY experiments with a representative hexamer suggested the presence of a 7/8 helical conformation in solution. Biological evaluation revealed a significant impact of the peptide chain length and the N-terminal cap on the antimicrobial and anticancer properties of this series of hybrid peptides. The Fmoc-capped peptide 6e displayed the most potent antimicrobial activity against a panel of Gram-negative and Gram-positive bacterial strains (e. g. against E. Coli: MIC=8 mg/L; S. aureus: MIC=4 mg/L).

Strain-promoted azide-alkyne cycloadditions (SPAAC) have proven extremely useful for labeling of biomolecules, but typically produce isomeric mixtures. This is not appropriate for the formation of bioactive molecules in living cells. Here, the first use of SPAAC for the isomer-free synthesis of a bioactive molecule is reported both in vitro and inside cultured cells. We developed the symmetrical cyclooctyne SYPCO and used it for the generation of a chemically uniform triazole inhibitor of protein-protein interactions mediated by Bcl-xL via isomer-free SPAAC (iSPAAC). Tumor cells treated with the reactants of the iSPAAC reaction contained higher concentrations of triazole, and displayed higher apoptosis levels, than cells treated with pre-synthesized triazole. We envision iSPAAC as a broadly applicable method for modulating intracellular targets with organic molecules with molecular weights prohibitively large for cellular uptake, via smaller and thus more cell-permeable components.

Aims High levels of glucose and reactive carbonyl intermediates of its degradation pathway such as methylglyoxal (MG) may contribute to diabetic complications partly via increased generation of reactive oxygen species (ROS). This study focused on glutathione peroxidase-1 (GPx1) expression and the impact of carbonylation as an oxidative protein modification on GPx1 abundance and activity in human umbilical vein endothelial cells (HUVEC) under conditions of mild to moderate oxidative stress. Results High extracellular glucose and MG enhanced intracellular ROS formation in HUVECs. Protein carbonylation was only transiently augmented pointing to an effective antioxidant defense in these cells. Nitric oxide synthase expression was decreased under hyperglycemic conditions but increased upon exposure to MG, whereas superoxide dismutase expression was not significantly affected. Increased glutathione peroxidase (GPx) activity seemed to compensate for a decrease in GPx1 protein due to enhanced degradation via the proteasome. Mass spectrometry analysis identified Lys-114 as a possible carbonylation target which provides a vestibule for the substrate H2O2 and thus enhances the enzymatic reaction. Innovation Oxidative protein carbonylation has so far been associated with functional inactivation of modified target proteins mainly contributing to aging and age-related diseases. Here, we demonstrate that mild oxidative stress and subsequent carbonylation seem to activate protective cellular redox signaling pathways whereas severe oxidative stress overwhelms the cellular antioxidant defense leading to cell damage. Conclusions This study may contribute to a better understanding of redox homeostasis and its role in the development of diabetes and related vascular complications., Graphical abstract fx1

A special domain of the thylakoid membrane of higher plants has been isolated which carries out the de-epoxidation of the xanthophyll cycle pigment violaxanthin to zeaxanthin. Recent models indicate that in the chloroplast of higher plants, the violaxanthin (V) cycle takes place within specialized domains in the thylakoid membrane. Here, we describe a new procedure to directly isolate such a domain in functional state. The procedure consists of a thylakoid membrane isolation at a pH value of 5.2 which realizes the binding of the enzyme V de-epoxidase (VDE) to the membrane throughout the preparation process. Isolated thylakoid membranes are then solubilized with the very mild detergent n-dodecyl α-D-maltoside and the pigment-protein complexes are separated by sucrose gradient ultracentrifugation. The upper main fraction of the sucrose gradient represents a V cycle domain which consists of the major light-harvesting complex of photosystem II (LHCII), a special lipid composition with an enrichment of the galactolipid monogalactosyldiacylglycerol (MGDG) and the VDE. The domain is isolated in functional state as evidenced by the ability to convert the LHCII-associated V to zeaxanthin. The direct isolation of a V cycle domain proves the most important hypotheses concerning the de-epoxidation reaction in intact thylakoid membranes. It shows that the VDE binds to the thylakoid membrane at low pH values of the thylakoid lumen, that it binds to membrane regions enriched in LHCII, and that the domain contains high amounts of MGDG. The last point is in line with the importance of the galactolipid for V solubilisation and, by providing inverted hexagonal lipid structures, for VDE activity.

The bacterial protein DnaK promotes folding of newly synthesized polypeptide chains, refolding of misfolded proteins, and protein trafficking. Assisted refolding is especially important under stress conditions induced by antibiotic therapies reducing the desired bactericidal effects. DnaK is supposedly targeted by proline-rich antimicrobial peptides (PrAMPs), but Escherichia coli ΔdnaK mutants and wild type strains are equally susceptible indicating further intracellular targets, such as the 70S ribosome. Crystal structures of PrAMPDnaK- complexes revealed forward and reverse binding modes at the substrate binding domain. Here, we used these ligand-target structures for the first time to rationally optimize peptides using molecular modeling and docking leading to the prediction of four-residue long sequences for improved binding to DnaK. When these sequences were used to replace the original sequence stretch in Onc72, most peptides showed significantly reduced dissociation constants (Kd) determined by fluorescence polarization. In a second approach, the X-ray structures of Api88 and Onc72 bound to DnaK were examined to predict substitutions prone to stronger interactions. Among the 36 peptides obtained from both approaches, six derivatives bound to DnaK with more than 10-fold higher affinities (Kd values in the low micromolar to nanomolar range). Peptides binding stronger to DnaK showed the same minimal inhibitory concentrations against wild type E. coli as the original peptide, but were slightly less active for ΔdnaK mutants. However, one peptide was able to overcome the resistance in an E. coli mutant lacking the SbmA transporter obligatory for the uptake of PrAMPs including Api88 and Onc72. Thus, it´s tempting to speculate that DnaK might be involved in the translocation of PrAMPs into E. coli.

Recent surveillance data on antimicrobial resistance predict the beginning of the post-antibiotic era with pan-resistant bacteria even overcoming polymyxin as the last available treatment option. Thus, new substances using novel modes of antimicrobial action are urgently needed to reduce this health threat. Antimicrobial peptides are part of the innate immune system of most vertebrates and invertebrates and accepted as valid substances for antibiotic drug development efforts. Especially, short proline-rich antimicrobial peptides (PrAMP) of insect origin have been optimized for activity against Gram-negative strains. They inhibit protein expression in bacteria by blocking the 70S ribosome exit tunnel (oncocin-type) or the assembly of the 50S subunit (apidaecin-type binding). Thus, apidaecin analog Api137 and oncocin analog Onc112 supposedly bind to different nearby or possibly partially overlapping binding sites. Here, we synthesized Api137/Onc112-conjugates bridged by ethylene glycol spacers of different length to probe synergistic activities and binding modes. Indeed, the antimicrobial activities against Escherichia coli and Pseudomonas aeruginosa improved for some constructs, although the conjugates did not bind better to the 70S ribosome of E. coli than Api137 and Onc112 using 5(6)-carboxyfluorescein-labelled Api137 and Onc112 in a competitive fluorescence polarization assay. In conclusion, Api137/Onc112-conjugates showed increased antimicrobial activities against P. aeruginosa and PrAMP-susceptible and -resistant E. coli most likely because of improved membrane interactions, whereas the interaction to the 70S ribosome was most likely not improved relying still on the independent apidaecin- and oncocin-type binding modes. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Reactive oxygen species (ROS) can oxidize virtually all cellular components. In proteins cysteine, methionine, tryptophan, and tyrosine residues are most prone to oxidation and their oxidized forms are thus considered as biomarkers of oxidative protein damages. Ultraviolet radiation and some endogenous ROS can produce tyrosine radicals reacting with other tyrosine residues yielding intra- or intermolecular cross-links in proteins. These 3,3'-dityrosines can be quantified by their characteristic fluorescence, but analytical methods to identify the modification sites in proteins are still missing. Although mass spectrometry (MS) is routinely used to map other post-translational modifications, the analysis of dityrosines is challenged by simultaneous fragmentations of both cross-linked peptide chains producing complex tandem mass spectra. Additionally, the fragmentation patterns differ from linear peptides. Here, we studied the fragmentation behavior of dityrosine cross-linked peptides obtained by incubating three peptides (AAVYHHFISDGVR, TEVSSNHVLIYLDK, and LVAYYTLIGASGQR) with horseradish peroxidase in the presence of hydrogen peroxide. Homo- and hetero-dimerization via dityrosine was monitored by fluorescence spectroscopy and MS. The fragmentation characteristics of dityrosine-linked peptides were studied on an ESI-LTQ-Orbitrap-MS using collision induced dissociation, which allowed localizing the cross-linked positions and provided generic rules to identify this oxidative modification. When human serum albumin oxidized with 50-fold molar excess of HOCl in phosphate buffer saline was analyzed by nanoRPC-ESI-MS/MS, an automatic database search considering all possible (in-silico generated) tyrosine-containing peptides as dynamic modifications revealed four different types of oxidatively modified tyrosine residues including dityrosines linking ten different Tyr residues. The automatic database search was confirmed by manual interpretation of each tandem mass spectrum.

Scope Thermal processing of soy milk kills pathogens and denatures anti-nutrition factors warranting microbiological safety, better digestibility, and longer storage. Additionally, Maillard reactions are triggered, yielding glycated proteins (Amadori/Heyns products) and a heterogeneous group of advanced glycation end-products (AGEs). These modifications alter the nutritional value, antigenicity, and digestibility of proteins. They also raise concerns about potentially toxic effects. This study aims at characterizing these modifications in proteins from different soy milk products. Methods and results Here, glycation and AGE-modification sites in the proteome of ultrahigh-temperature-treated natural soy milk, soy milk sweetened with hexose (fructose)-containing sweeteners (SSM), and sucrose as well as soy-based infant formulas (SIFs) from different manufacturers are reported for the first time. A bottom-up proteomic approach based on nano reversed-phase high-perfomance liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoRP-HPLC-ESI-MS/MS) (collision-induced dissociation (CID) and electron transfer dissociation modes) identified 229 glycated peptides and 128 AGE-modified peptides resembling 53 proteins. The glycation sites are mainly derived from hexoses, whereas Nδ -carboxyethylarginine and methylglyoxal-derived hydroimidazolone are the main AGEs in soy milk. Conclusion The qualitative and quantitative data obtained here indicate that early glycation increases with harsher processing conditions (SIFs) and the addition of hexose-containing sweeteners (SSMs), whereas the latter sweeteners (but not the harsher processing) triggered more AGE modifications.

19 Pags.- 2 Tabls.- 11 Figs.- Supp. Data (15 Figs.- 5 Tabls.- 4 DataSets). Copyright © 2018 American Society of Plant Biologists. All rights reserved. Authors cannot archive post-print (ie final draft post-refereeing): Must link to publisher version, toll-free link provided., Nitrogen fixation is an agronomically and environmentally important process catalyzed by bacterial nitrogenase within legume root nodules. These unique symbiotic organs have high metabolic rates and produce large amounts of reactive oxygen species that may modify proteins irreversibly. Here, we examined two types of oxidative posttranslational modifications of nodule proteins: carbonylation, which occurs by direct oxidation of certain amino acids or by interaction with reactive aldehydes arising from cell membrane lipid peroxides; and glycation, which results from the reaction of lysine and arginine residues with reducing sugars or their autooxidation products. We used a strategy based on the enrichment of carbonylated peptides by affinity chromatography followed by liquid chromatography-tandem mass spectrometry to identify 369 oxidized proteins in bean (Phaseolus vulgaris) nodules. Of these, 238 corresponded to plant proteins and 131 to bacterial proteins. Lipid peroxidation products induced most carbonylation sites. This study also revealed that carbonylation has major effects on two key nodule proteins. Metal-catalyzed oxidation caused the inactivation of malate dehydrogenase and the aggregation of leghemoglobin. In addition, numerous glycated proteins were identified in vivo, including three key nodule proteins: sucrose synthase, glutamine synthetase, and glutamate synthase. Label-free quantification identified 10 plant proteins and 18 bacterial proteins as age-specifically glycated. Overall, our results suggest that the selective carbonylation or glycation of crucial proteins involved in nitrogen metabolism, transcriptional regulation, and signaling may constitute a mechanism to control cell metabolism and nodule senescence., This work was supported by the Ministerio de Economía y Competitividad-Fondos Europeos de Desarrollo Regional (grant AGL2014-53717-R to M.A.M. and M.B.), by the German Research Society (grant FR3117/2-1 to A.F. and A.K.), by the Russian Foundation for Basic Research (grant 18-016-00190 to A.F.), by the Deutsche Forschungsgemeinschaft (grant FE-1236/3-1 to M.F.), and by the European Regional Development Fund (European Union and Free State Saxony; grants 100146238 and 100121468 to M.F.).

Polo-like kinase 1 (Plk1), a validated cancer target, harbors a protein-protein interaction domain referred to as the polo-box domain (PBD), in addition to its enzymatic domain. Although functional inhibition either of the enzymatic domain or of the PBD has been shown to inhibit Plk1, so far there have been no reports of bifunctional agents with the potential to target both protein domains. Here we report the development of Plk1 inhibitors that incorporate both an ATP-competitive ligand of the enzymatic domain, derived from BI 2536, and a functional inhibitor of the PBD, based either on the small molecule poloxin-2 or on a PBD-binding peptide. Although these bifunctional agents do not seem to bind both protein domains simultaneously, the most potent compound displays low-nanomolar activity against the Plk1 PBD, with excellent selectivity over the PBDs of Plk2 and Plk3. Our data provide insights into challenges and opportunities relating to the optimization of Plk1 PBD ligands as potent Plk1 inhibitors.

Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid-protein adducts by liquid chromatography-MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid-peptide adducts generated by incubating model peptides corresponding to the amphipathic β1 sheet sequence of apolipoprotein B-100 with 1-palmitoyl-2-(oxo-nonanoyl)-sn-glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide-lipid adducts was separated by TWIMS, which was especially important for the identification of two mono-PONPC-peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide-lipid adduct possessing most likely different orientations of the hydrophobic sn-1 fatty acyl residue and head group of PONPC, relative to the peptide backbone.

Gene-encoded antimicrobial peptides (AMPs) kill bacteria very efficiently by either lytic mechanisms or inhibition of specific bacterial targets. Proline-rich AMPs (PrAMPs), for example, produced in insects and mammals rely on the second mechanism. They bind to the 70 kDa bacterial heat shock protein DnaK and the 60 kDa chaperonin GroEL and interfere with protein folding, but this does not explain their strong bactericidal effects. Thus, we looked for further binding partners of apidaecin 1b, originally identified in honey bees, and two rationally optimized analogues (Api88 and Api137). Because affinity chromatography using Api88 as an immobilized ligand enriched only a few proteins at low levels besides DnaK, we synthesized Api88 analogues substituting Tyr7 with p-benzoyl-phenylalanine (Bpa), which can cross-link the peptide to binding partners after UV irradiation. Escherichia coli was incubated with biotinylated Api88 Tyr7Bpa or the corresponding all-d-peptide, irradiated, and lysed. The protein extract was enriched by streptavidin, separated by SDS-PAGE, digested with trypsin, and analyzed by nanoRP-UPLC-ESI-QqTOF-MS/MS. Among the 41 proteins identified, 34 were detected only in the l-Api88 Tyr7Bpa sample, including five 70S ribosomal proteins, DNA-directed RNA polymerase, and pyruvate dehydrogenase, indicating that PrAMPs might interfere with protein translation and energy metabolism.

Oxysterol species are formed in vivo by enzymatic and non-enzymatic oxidation of cholesterol. Oxysterols were shown not only to be intermediates in the biosynthesis of bile acids and steroid hormones but also to possess versatile bioactivities. Many functions of oxysterols are not fully understood, others may not have been discovered yet, especially those of non-enzymatic origin. The limited accessibility to standard compounds challenges both analytics and functional studies. Here an amperometric flow-through electrochemical (EC) oxidation of cholesterol was applied to generate numerous oxidation products within short reaction times. Besides nine oxysterols confirmed with standard compounds (LC-MS), more than 10 additional, not identified oxidation products were present. The oxidation sites of the identified compounds were in agreement to the expected accessibility of the cholesterol backbone to free radical driven oxidation. Additionally, the presence of three products which are known to be synthesized enzymatically in vivo demonstrated a successful mimicking of these processes by EC as well. Several of the unidentified species showed the same analytical behavior (retention time and MS/MS) as compounds observed in extracts from a cardiomyocyte cell model of nitrosative stress. Further investigation of electrochemically generated compounds will allow identification and characterization of new oxysterols in vivo. Practical applications: Electrochemical oxidation of cholesterol generates numerous oxysterols in short reaction times. The identified products were oxysterols known to be generated in vivo by both free radical and enzymatic processes. Among the unidentified oxidation products were species which showed the same analytical behavior as substances in extracts from stressed cell cultures. Further investigation of EC-generated compounds may facilitate identification and characterization of new in vivo relevant oxysterols and the synthesis of standards for biological and analytical applications. Electrochemical oxidation of cholesterol generates numerous oxysterols in short reaction times. The nine identified products were oxysterols known to be generated in vivo by both free radical and enzymatic processes. Among the 15 unidentified oxidation products were species which showed the same analytical behavior as substances in extracts from stressed cell-cultures, indicating biological significance of the new electrochemically generated oxysterols.

Adiponectin, an adipose tissue-excreted adipokine plays protective roles in metabolic and cardiovascular diseases and exerts anti-cancer activities, partially by interfering with leptin-induced signaling. Previously we identified the active site in the adiponectin protein, and generated both a nanomolar monomeric agonist of the adiponectin receptor (10-mer ADP355) and an antagonist (8-mer ADP400) to modulate various adiponectin receptor-mediated cellular functions. As physiologically circulating adiponectin forms multimeric complexes, we also generated an agonist dimer with improved biodistribution and in vitro efficacy. In the current report, we attempted to optimize the monomeric agonist structure. Neither extension of the peptide up to 14-mer analogs nor reinstallation of native residues in permissible positions enhanced significantly the activity profile. The only substitutions that resulted in 5-10-fold improved agonistic activity were the replacement of turn-forming Gly4 and Tyr7 residues with Pro and Hyp, respectively, yielding the more active native β-sheet structure. All peptides retained good stability in human serum exhibiting half-lives >2 h. The cellular efficacy and stability rankings among the peptides followed expected structure-activity relationship trends. To investigate whether simultaneous activation of adiponectin pathways and inhibition of leptin-induced signals can result in cytostatic and anti-oncogenic signal transduction processes, we developed a chimera of the leptin receptor antagonist peptide Allo-aca (placed to the N-terminus) and ADP355 (at the C-terminus). The in vitro anti-tumor activity and intracellular signaling of the chimera were dominated by the more active Allo-aca component. The ADP355 part, however, reversed unfavorable in vivo metabolic effects of the leptin receptor antagonist.

Agonists of the glucagon-like peptide-1 (GLP-1) receptor and analogs of human amylin have been studied for almost two decades due to their therapeutic potential to treat diabetes mellitus and obesity. Both native peptides exhibit unfavorable pharmacokinetics. Even optimized analogs less prone to proteolysis have to be applied at least daily or once-weekly utilizing microsphere formulations or fusion to proteins. Thus, innovative approaches allowing tuning the drug levels to achieve beneficial therapeutic responses and prolonged application intervals are demanded. PEGylation, i.e., conjugation of polyethylene glycol (PEG), has enhanced the bioavailability of several drugs but does not appear to be useful for amylin and GLP-1. Thus, we developed a traceless prodrug strategy using protease-cleavable peptide linkers that can release therapeutic peptides. Specifically, the release kinetics of linker sequences LVPR, LDPR, and LVPRLVPR were tested in combination with GLP-1 analog taspoglutide, amylin, and amylin analog pramlintide in mouse serum. The linkers allowed tuning the taspoglutide release over more than one order of magnitude providing stable serum levels from ~0.08 to 3 μmol/L for ~20 h. Amylin and pramlintide levels were ~20 nmol/L and stable for at least 24 h. Importantly, all peptide therapeutics were protected against proteolytic degradation within the prodrug, especially the N-terminal sequences near the PEG. Thus, taspoglutide was released even after an incubation period of 24 h in serum with the content of degraded taspoglutide being below 2% in the prodrug at this time point. This PEG-prodrug technology could provide precisely tuned long-acting anti-diabetic and anti-obesity therapies and even once-monthly administration intervals when combined with other formulation strategies.

The recent upsurge of multidrug resistant bacteria (MDRB) among global communities has become one of the most serious challenges facing health professionals and the human population worldwide. Cationic ultrashort antimicrobial peptides (USAMPs) are a promising group of molecules that meet the required criteria of novel antimicrobial drug development. UP-5, a novel penta-peptide, displayed significant antimicrobial activities against various standard and clinical isolates of MDRB. UP-5 displayed MICs values within the range of (10-15 μM) and (55-65 μM) against Gram-positive and Gram-negative bacteria, respectively. Furthermore, UP-5 displayed antibiofilm activity with minimum biofilm eradication concentration (MBEC) value as equal to twofold higher than MIC value. At the same inhibitory concentrations, UP-5 exhibited very low or negligible toxicity toward human erythrocytes and mammalian cells. Combining UP-5 with conventional antibiotics led to a synergistic or additive mode of action that resulted in the reduction of the MIC values for some of the antibiotics by 99.7% along a significant drop in MIC values of the peptide. The stability profile of UP-5 was evaluated in full mouse plasma and serum with results indicating a more stable pattern in plasma. The present study indicates that USAMPs are promising antimicrobial agents that can avoid the negative characteristics of conventional antimicrobial peptides. Additionally, USAMPs exhibit good to moderate activity against MDRB, negligible toxicity, and synergistic outcomes in combination with conventional antimicrobial agents.

Kefir has a long tradition in human nutrition due to its presupposed health promoting effects. To investigate the potential contribution of bioactive peptides to the physiological effects of kefir, comprehensive analysis of the peptide profile was performed by nano-ESI-LTQ-Orbitrap MS coupled to nano-ultrahigh-performance liquid chromatography. Thus, 257 peptides were identified, mainly released from ß-casein, followed by ?S1-, ?-, and ?S2-casein. Most (236) peptides were uniquely detected in kefir, but not in raw milk indicating that the fermentation step does not only increase the proteolytic activity 1.7- to 2.4-fold compared to unfermented milk, but also alters the composition of the peptide fraction. The influence of the microflora was determined by analyzing kefir produced from traditional kefir grains or commercial starter culture. Kefir from starter culture featured 230 peptide sequences and showed a significantly, 1.4-fold higher proteolytic activity than kefir from kefir grains with 127 peptides. A match of 97 peptides in both varieties indicates the presence of a typical kefir peptide profile that is not influenced by the individual composition of the microflora. Sixteen of the newly identified peptides were previously described as bioactive, including angiotensin-converting enzyme (ACE)-inhibitory, antimicrobial, immunomodulating, opioid, mineral binding, antioxidant, and antithrombotic effects. Biological significance: The present study describes a comprehensive peptide profile of kefir comprising 257 sequences. The peptide list was used to identify 16 bioactive peptides with ACE-inhibitory, antioxidant, antithrombotic, mineral binding, antimicrobial, immunomodulating and opioid activity in kefir. Furthermore, it was shown that a majority of the kefir peptides were not endogenously present in the raw material milk, but were released from milk caseins by proteases of the microbiota and are therefore specific for the product. Consequently, the proteolytic activity and the composition of the peptide profile can be controlled by the applied microflora (grains or starter culture). On the other hand, a considerable portion of the peptide profile was identified to be typical for kefir in general and independent from production parameters.In summary, the generated kefir peptide profile helped to reveal its origin and to identify bioactive peptides in kefir, which may advance the understanding of health benefits of this food product. The results further indicate that subsets of the kefir peptide list can be used as markers to control food authenticity, for example, to distinguish different types of kefir. © 2015 Elsevier B.V., Türkiye Bilimsel ve Teknolojik Araştirma Kurumu: 1100797 01DL12033, The present study was funded by the Intensified Cooperation Program (IntenC) for the Promotion of German-Turkish Higher Education Research of the International Bureau of the German Federal Ministry of Education and Research (grant No. 01DL12033 ) and Turkey's Scientific and Technological Research Council (TÜBITAK, grant No. 1100797 ). The funders were not involved in study design, the collection, analysis and interpretation of data, writing the report or in the decision to submit the article for publication.

Glycation, or non-enzymatic glycosylation, is a common protein modification formed by reactions between reducing sugars (i.e. aldoses and ketoses) with protein amino groups. Resulting Amadori and Heyns compounds, respectively, can be oxidatively degraded yielding a structurally heterogeneous group of advanced glycation end-products. We have studied this process in aqueous conditions at 95 °C in terms of appearing products and their formation kinetics in the presence or absence of reactive oxygen species (ROS)-generating systems (iron(II) sulfate). RP-HPLC-ESI-MS revealed 20 products, 12 of which were confirmed after synthesis by identical retention times and fragmentation patterns. These products accumulated during the incubation period of 4 h (N(ε)-carboxymethyl-, N(ε)-formyl- and N(ε)-methyl lysine) or appeared intermediately (2-aminoadipic semialdehyde, N(ε)-ethanalyl lysine). Acidic and basic amino acid residues near the glycation site and elevated ROS levels in the reaction mixture had significant effects on both product formation and degradation kinetics.

Proline-rich antimicrobial peptides (PrAMPs) freely penetrate through the outer membrane into the periplasm of Gram-negative bacteria, before they are actively translocated by a permease/transporter-mediated uptake into the cytoplasm where they are reported to inhibit chaperone DnaK. Here we have studied the PrAMP apidaecin 1b, which is produced in honey bees in response to bacterial infections, and optimized apidaecin analogs for their bacterial uptake. The peptides were labeled with 5(6)-carboxyfluorescein and their internalization in Escherichia coli and Klebsiella pneumoniae was visualized by fluorescence microscopy and quantified by flow cytometry for four different time points over an incubation period of 4 h. Apidaecin 1b entered only 40% to 50% of the cells at detectable quantities, whereas designer peptides Api88, Api134 and Api155 entered more than 95% of the bacteria within 30 min at around fourfold higher quantities than the native peptide. Interestingly, a shortened version designated as (1-17)Api88, bound DnaK as efficiently as the 18-residue long Api88 and entered the bacteria at similar kinetics as Api88, but was unable to inhibit the bacterial growth. Similar conflicts with currently proposed mechanisms of PrAMPs were also obtained for some Ala-substituted analogs and reverse apidaecin sequences. Although peptides with C-terminal amides enter the cells much more efficiently than homologous C-terminal acids, this improved cell penetration does not improve the antibacterial activities. These studies suggest that PrAMPs utilize additional modes of action to kill sensitive organisms.

Optical tweezers-assisted dynamic force spectroscopy is employed to investigate specific receptor-ligand interactions on the level of single binding events. In particular, we analyze binding of the phosphorylation-specific monoclonal antibody (mAb) HPT-101 to synthetic tau-peptides with two potential phosphorylation sites (Thr231 and Ser235), being the most probable markers for Alzheimer's disease. Whereas the typical interpretation of enzyme-linked immunosorbent assay (ELISA) suggests that this monoclonal antibody binds exclusively to the double-phosphorylated tau-peptide, we show here by DFS that the specificity of only mAb HPT-101 is apparent. In fact, binding occurs also to each sort of monophosphorylated peptide. Therefore, we characterize the unbinding process by analyzing the measured rupture force distributions, from which the lifetime of the bond without force τ0, its characteristic length xts, and the free energy of activation ΔG are extracted for the three mAb/peptide combinations. This information is used to build a simple theoretical model to predict features of the unbinding process for the double-phosphorylated peptide purely based on data on the monophosphorylated ones. Finally, we introduce a method to combine binding and unbinding measurements to estimate the relative affinity of the bonds. The values obtained for this quantity are in accordance with ELISA, showing how DFS can offer important insights about the dynamic binding process that are not accessible with this common and widespread assay.

Ribonuclease S (RNase S) is a versatile model system to introduce non-canonical amino acids into a protein scaffold by self-assembly of the protein complex from S-protein and S-peptide. We have introduced two cysteines into the S-peptide replacing lysine-7 and glutamine-11 of the α-helical region of this peptide. The crystal structure of this variant was determined in the apo state and in complex with HgII ions. These structures serve as a template for the construction of artificial hybrid catalysts based on RNase S, e.g. for phosphanyl amino acids complexing transition metal ions for organometallic catalysis.

Hsp70 chaperones have been implicated in assisting protein folding of newly synthesized polypeptide chains, refolding of misfolded proteins, and protein trafficking. For these functions, the chaperones need to exhibit a significant promiscuity in binding to different sequences of hydrophobic peptide stretches. To characterize the structural basis of sequence specificity and flexibility of the Escherichia coli Hsp70 chaperone DnaK, we have analyzed crystal structures of the substrate binding domain of the protein in complex with artificially designed peptides as well as small proline-rich antimicrobial peptides. The latter peptides from mammals and insects were identified to target DnaK after cell penetration. Interestingly, the complex crystal structures reveal two different peptide binding modes. The peptides can bind either in a forward or in a reverse direction to the conventional substrate binding cleft of DnaK in an extended conformation. Superposition of the two binding modes shows a remarkable similarity in the side chain orientations and hydrogen bonding pattern despite the reversed peptide orientation. The DnaK chaperone has evolved to bind peptides in both orientations in the substrate binding cleft with comparable energy without rearrangements of the protein. Optimal hydrophobic interactions with binding pockets -2 to 0 appear to be the main determinant for the orientation and sequence position of peptide binding.

Increasing death tolls accounted for by antimicrobial drug resistance demand novel antibiotic lead compounds. Among different promising candidate classes, proline-rich antimicrobial peptides (PrAMPs) are very favorable due to their intracellular mechanism, i.e., binding to the 70S ribosome and DnaK, after active uptake relying on bacterial transporters like SbmA and MdtM. Studies on peptide internalization as the first step of their complex mode of action rely typically on fluorophore or radioactive labeling and quantification using microscopy, flow cytometry, or radioactivity. Here, a liquid chromatography based assay was applied to quantify the unlabeled internalized full-length peptides and their proteolytic degradation products (metabolites) using UV absorbance and mass spectrometry. Knockout mutants lacking transporter proteins showed reduced PrAMP uptakes, explaining their reduced susceptibility against PrAMPs. Interestingly, major metabolites produced by bacterial proteases still bound to the 70S ribosome provide evidence that degradation by cytosolic proteases as a possible resistance mechanism is not very efficient. Graphical abstract The uptake of unlabeled proline-rich antimicrobial peptides (PrAMPs) is analyzed in Escherichia coli BW25113 wild-type and transporter knockout mutants ΔsbmA and BS2 (ΔsbmA yjiL::Tn10) by reversed-phase chromatography and quantified by UV detection or mass spectrometry with multi-reaction monitoring (scheme right). Internalized peptide amounts correlated to minimal inhibitory concentrations and bacterial transport activities based on the present transporter proteins (scheme left).

During thermal treatment of milk, proteins are oxidized, which may reduce the nutritional value of milk, abolish protein functions supporting human health, especially important for newborns, and yield potentially harmful products. The side chains of several amino acids can be oxidized to reactive carbonyls, which are often used to monitor oxidative stress in organisms. Here we mapped protein carbonylation sites in raw milk and different brands of pasteurized, ultra high temperature (UHT) treated milk, and infant formulas (IFs) after digesting the precipitated proteins with trypsin. Reactive carbonyls were derivatized with O-(biotinylcarbazoylmethyl)hydroxylamine to enrich the modified peptides by avidin-biotin affinity chromatography and analyze them by nanoRP-UPLC-ESI-MS. Overall, 53 unique carbonylated peptides (37 carbonylation sites, 15 proteins) were identified. Most carbonyls were derived from dicarbonyls (mainly glyoxal). The number of carbonylation sites increased with the harsher processing from raw milk (4) to pasteurized (16) and UHT milk (16) and to IF (24).

Proline-rich antimicrobial peptides (PrAMPs) from insects and mammals have recently been evaluated for their pharmaceutical potential in treating systemic bacterial infections. Besides the native peptides, several shortened, modified, or even artificial sequences were highly effective in different murine infection models. Most recently, we showed that the 18-residue-long peptide Api88, an optimized version of apidaecin 1b, was efficient in two different animal infection models using the pathogenic Escherichia coli strains ATCC 25922 and Neumann, with a promising safety margin. Here, we show that Api88 is degraded relatively fast upon incubation with mouse serum, by cleavage of the C-terminal leucine residue. To improve its in vitro characteristics, we aimed to improve its serum stability. Replacing the C-terminal amide by the free acid or substituting Arg-17 with l -ornithine or l -homoarginine increased the serum stabilities by more than 20-fold (half-life, ∼4 to 6 h). These analogs were nontoxic to human embryonic kidney (HEK 293), human hepatoma (HepG2), SH-SY5Y, and HeLa cells and nonhemolytic to human erythrocytes. The binding constants of all three analogs with the chaperone DnaK, which is proposed as the bacterial target of PrAMPs, were very similar to that of Api88. Of all the analogs tested, Api137 (Gu-ONNRPVYIPRPRPPHPRL; Gu is N , N , N ′, N ′-tetramethylguanidino) appeared most promising due to its high antibacterial activity, which was very similar to Api88. Positional alanine and d -amino acid scans of Api137 indicated that substitutions of residues 1 to 13 had only minor effects on the activity against an E. coli strain, whereas substitutions of residues 14 to 18 decreased the activity dramatically. Based on the significantly improved resistance to proteolysis, Api137 appears to be a very promising lead compound that should be even more efficient in vivo than Api88.

The fragmentation behavior of the 2+ and 3+ charge states of eleven different phosphorylated tau peptides was studied using collision-induced dissociation (CID), electron transfer dissociation (ETD) and metastable atom-activated dissociation (MAD). The synthetic peptides studied contain up to two known phosphorylation sites on serine or threonine residues, at least two basic residues, and between four and eight potential sites of phosphorylation. CID produced mainly b-/y-type ions with abundant neutral losses of the phosphorylation modification. ETD produced c-/z-type ions in highest abundance but also showed numerous y-type ions at a frequency about 50% that of the z-type ions. The major peaks observed in the ETD spectra correspond to the charge-reduced product ions and small neutral losses from the charge-reduced peaks. ETD of the 2+ charge state of each peptide generally produced fewer backbone cleavages than the 3+ charge state, consistent with previous reports. Regardless of charge state, MAD achieved more extensive backbone cleavage than CID or ETD, while retaining the modification(s) in most cases. In all but one case, unambiguous modification site determination was achieved with MAD. MAD produced 15–20% better sequence coverage than CID and ETD for both the 2+ and 3+ charge states and very different fragmentation products indicating that the mechanism of fragmentation in MAD is unique and complementary to CID and ETD. Copyright © 2012 John Wiley & Sons, Ltd.

The specific interaction of peptides with proteins is often a key factor which determines biological activities. The determination of K d values of such interactions is commonly performed with fluorescence polarization. However, fluorescence polarization assays are prone to false-positive results due to the potential for non-specific interactions and only afford very low signal-to-background ratios. Here, we present as an alternative a fluorescence resonance energy transfer based quenching assay to measure peptide–protein interactions in solution. In a test setup where antimicrobial peptides were tested for their affinity towards the protein DnaK, the assay provided high specificity and good reproducibility and correlated with the results obtained by fluorescence polarization methods. Furthermore, we established a fast prescreening method which will allow a highly efficient screening of peptide libraries by reducing the amount of sample by 98 % compared to conventional fluorescence polarization assays.

Reactive oxygen species (ROS) can oxidize proteins at almost any amino acid residue. Whereas some modifications are reversible within the cells, the higher oxidation states are especially irreversible. These irreversible post translational modifications are widely used as biomarkers of oxidative stress, such as protein carbonylation, which refers to aldehydes, ketones and lactams as ‘reactive carbonyl groups’. This study relied on a set of synthetic peptides containing a C-terminal aldehyde (arginal) or modification with pyruvic acid (ketone) or 4-hydroxynonenal (aldehyde) at lysine or histidine residues, as well as peptides containing pyroglutamic acid (oxidation product of proline) and 2-amino-3-butyric acid (oxidation product of threonine). The carbonylation sites were specifically derivatized with 2,4-dinitrophenylhydrazine (DNPH) and the fragmentation behavior of the products investigated in electrospray ionization (ESI-) MS. Importantly, the DNPH-labeled carbonylated peptides showed favorable ionization behaviors in negative ion mode ESI, providing a sensitive detection method. Regular peptides were mostly discriminated under these conditions. Among the fragmentation techniques tested for the negatively charged ions, pulsed Q dissociation provided three diagnostic ions at m/z values 152.0, 163.1 and 179.0, specific for DNPH-modified peptides. These marker ions were successfully applied to detect the carbonylated model peptides in a spiked tryptic digest of bovine serum albumin and a complex protein mixture obtained from HeLa cells.

Proteases are widely used in analytical sciences and play a central role in several widespread diseases. Thus, there is an immense need for highly adaptable and sensitive assays for the detection and monitoring of various proteolytic enzymes. We established a simple protease fluorescence resonance energy transfer (pro-FRET) assay for the determination of protease activities, which could in principle be adapted for the detection of all proteases. As proof of principle, we demonstrated the potential of our method using trypsin and enteropeptidase in complex biological mixtures. Briefly, the assay is based on the cleavage of a FRET peptide substrate, which results in a dramatic increase of the donor fluorescence. The assay was highly sensitive and fast for both proteases. The detection limits for trypsin and enteropeptidase in Escherichia coli lysate were 100 and 10 amol, respectively. The improved sensitivity for enteropeptidase was due to the application of an enzyme cascade, which leads to signal amplification. The pro-FRET assay is highly specific as even high concentrations of other proteases did not result in significant background signals. In conclusion, this sensitive and simple assay can be performed in complex biological mixtures and can be easily adapted to act as a versatile tool for the sensitive detection of proteases.

Ion mobility spectrometry (IMS), and particularly differential or field asymmetric waveform IMS (FAIMS), was recently shown capable of separating peptides with variant localization of post-translational modifications. However, that work was limited to a model peptide with Ser phosphorylation on fairly distant alternative sites. Here, we demonstrate that FAIMS (coupled to electrospray/mass spectrometry (ESI/MS)) can broadly baseline-resolve variant phosphopeptides from a biologically modified human protein, including those involving phosphorylation of different residues and adjacent sites that challenge existing tandem mass spectrometry (MS/MS) methods most. Singly and doubly phosphorylated variants can be resolved equally well and identified without dissociation, based on accurate separation properties. The spectra change little over a range of infusion solvent pH; hence, the present approach should be viable in conjunction with chromatographic separations using mobile phase gradients.

The proline-rich antimicrobial peptide oncocin is remarkably active in vitro against a number of important Gram-negative bacteria of concern to humans owing to their increasing resistance to antibiotics, i.e. Enterobacteriaceae ( Escherichia coli , Klebsiella pneumoniae and Enterobacter cloacae ) and non-fermenting species ( Acinetobacter baumannii and Pseudomonas aeruginosa ). Degradation of oncocin in mouse serum was investigated in this study. Several approaches to stabilise the main cleavage sites (C-terminal to Arg-15 and N-terminal to Arg-19) by substituting either or both arginine (Arg) residues with non-proteinogenic amino acids, i.e. α-amino-3-guanidino-propionic acid, homoarginine, nitro-arginine, N -methyl-arginine, β-homoarginine, d-arginine (d-Arg) or ornithine (Orn), were tested. These modifications were found to increase the half-life of oncocin in full mouse serum. For oncocin with two Orn residues in positions 15 and 19, the half-life in full serum increased from 25min to 3h. An increase of >8h was observed for oncocin with two d-Arg residues at these same positions. The antibacterial activities of these modified sequences were slightly better than the original oncocin sequence. Moreover, the three most stable analogues were found to be bactericidal against E. coli and were not toxic to HeLa cells or haemolytic to human erythrocytes.

ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5-HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the α-amino group of both amino acids, and the commercially available 5-HTP, with Fmoc-succinimide. High yields of Fmoc-Kyn were obtained by acid hydrolysis of Fmoc-NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M-type), and β-enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5-HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion-pair reagent on a C₁₈-phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan.

Glycation (or non-enzymatic glycosylation) is a common non-enzymatic covalent modification of human proteins. Glucose, the highest concentrated monosaccharide in blood, can reversibly react with amino groups of proteins to form Schiff bases that can rearrange to form relatively stable Amadori products. These can be further oxidized to advanced glycation end products (AGEs). Here, we analyzed the glycation patterns of human serum albumin (HSA) in plasma samples obtained from five patients with type 2 diabetes mellitus. Therefore, glycated peptides from a tryptic digest of plasma were enriched with m-aminophenylboronic acid (mAPBA) affinity chromatography. The glycated peptides were then further separated in the second dimension by RP-HPLC coupled on-line to an electrospray ionization (ESI) tandem mass spectrometer (MS/MS). Altogether, 18 Amadori peptides, encompassing 40% of the HSA sequence, were identified. The majority of the peptides were detected and relatively quantified in all five samples with a high reproducibility among the replicas. Eleven Lys-residues were glycated at similar quantities in all samples, with glycation site Lys(549) (K(Am(Glc))QTALVELVK) being the most abundant. In conclusion, the established mAPBA/nanoRP-HPLC-ESI-MS/MS approach could reproducibly identify and quantify glycation sites in plasma samples, potentially useful in diagnosis and therapeutic control.

Reactive oxidative species (ROS) play important roles in cellular signaling but can also modify and often functionally inactivate other biomolecules. Thus, cells have developed effective enzymatic and nonenzymatic strategies to scavenge ROS. However, under oxidative stress, ROS production is able to overwhelm the scavenging systems, increasing the levels of functionally impaired proteins. A major class of irreversible oxidative modifications is carbonylation, which refers to reactive carbonyl-groups. In this investigation, we have studied the production and clearance rates for skeletal muscle proteins in a rat model of acute oxidative stress over a time period of 24 h using a gel-based proteomics approach. Optimized ELISA and Western blots with 10-fold improved sensitivities showed that the carbonylation level was stable at 4 nmol per mg protein 3 h following ROS induction. The carbonylation level then increased 3-fold over 6 h and then remained stable. In total, the oxidative stress changed the steady state levels of 20 proteins and resulted in the carbonylation of 38 skeletal muscle proteins. Carbonylation of these proteins followed diverse kinetics with some proteins being highly carbonylated very quickly, whereas others peaked in the 9 h sample or continued to increase up to 24 h after oxidative stress was induced.

Increased levels of reactive oxygen species (ROS) cause oxidative stress and are believed to play a key role in the development of age-related diseases and mammalian aging in general by oxidizing proteins, lipids, and DNA. In this study, we have investigated the effects of ROS on actin in an established rat model of acute oxidative stress using short-term X-ray irradiation. Relative to the control, the actin functions studied in vitro were reduced for (i) actin polymerization to a minimum of 33% after 9 h and (ii) actin activated Mg(2+)-ATPase activity of myosin to 55% after 9 h. At 24 h, the activities had partially recovered to 64 and 80% of the control sample, respectively. The underlying oxidative modifications were also studied at the molecular level. The content of reactive carbonyl-groups increased 4-fold within the studied 24 h period. Among the five cysteine residues of actin, Cys(239) and Cys(259) were oxidized to sulfenic (Cys-SOH), sulfinic (Cys-SO(2)H), or sulfonic (Cys-SO(3)H) acids by increasing amounts over the time periods studied. The content of methionine sulfoxides also increased for 15 of the 16 methionine residues, with Met(44), Met(47), and Met(355) having the highest sulfoxide contents. Met(82) was also further oxidized to the sulfone. Among the four tryptophan residues present in actin, only Trp(79) and Trp(86) appeared to undergo oxidation. The relative contents of hydroxy-tryptophan, N-formyl-kynurenine, and kynurenine increased after irradiation, reaching a maximum in the 9 h sample.

Oxidative stress caused by an imbalance of the production of “reactive oxygen species” (ROS) and cellular scavenging systems is known to a play a key role in the development of various diseases and aging processes. Such elevated ROS levels can damage all components of cells, including proteins, lipids and DNA. Here, we study the influence of highly reactive ROS species on skeletal muscle proteins in a rat model of acute oxidative stress caused by X-ray irradiation at different time points. Protein preparations depleted for functional actin by polymerization were separated by gel electrophoresis in two dimensions by applying first non-reductive and then reductive conditions in SDS-PAGE. This diagonal redox SDS-PAGE revealed significant alterations to intra- and inter-molecular disulfide bridges for several proteins, but especially actin, creatine kinase and different isoforms of the myosin light chain. Though the levels of these reversible modifications were increased by oxidative stress, all proteins followed different kinetics. Moreover, a significant degree of protein was irreversibly oxidized (carbonylated), as revealed by western blot analyses performed at different time points.

We report here the first results of a method to extract and sequence mature enamel proteins from modern human and Neanderthal tooth enamel. Using MALDI-TOF/TOF mass spectrometry and a combination of direct sequencing and peptide mass mapping we have sequenced a peptide from the tyrosine-rich amelogenin peptide (TRAP) of the X isoform of the amelogenin protein for modern and recent human samples. We also report our results from two Neanderthal enamel samples where we were also able to recover fragments of the TRAP protein, which had a similar sequence to the modern human samples.

Apidaecins are 18–20-residue long proline-rich peptides expressed in insects as part of the innate immune system. They are very active against Gram-negative bacteria, especially Enterobacteriaceae. The C-terminal sequence PRPPHPRL is highly conserved, whereas the N-terminal region is variable. By replacing all 18 residues of apidaecin 1a and apidaecin 1b individually by alanine (Ala-scan), we have shown that single mutations in the C-terminal half of the peptides drastically reduced and mostly abolished the antibacterial activity against Escherichia coli. Conversely, substitutions in the N-terminal eight residues produced no, or only minor effects. The activity loss was correlated to the ability of apidaecin 1b and its mutants to enter Gram-negative bacteria, most likely because they no longer bind to a protein transporter. This assumed binding, however, was not inhibited by truncated apidaecin peptides added at tenfold higher concentrations. Interestingly, the antibacterial activity of full length apidaecin 1b was enhanced about four times by addition of a N-terminally truncated apidaecin peptide [11–18]-apidaecin 1b, as indicated by lower MIC-values against E. coli, although the short 5(6)-carboxyfluorescein-labeled peptide did not enter the bacteria. In contrast, the activity against the Gram-positive bacterium Micrococcus luteus was not located in the C-terminal sequence of apidaecins 1a and b, but depended mostly on the presence of all four basic residues.

Apidaecin peptides from Apis mellifera hemolymph are believed to attack intracellular bacterial targets. Our in vivo results for apidaecins 1a and 1b confirm that bacterial activity is non-lytic, however, the manner in which these peptides pass through the cell membrane to exert this activity is unknown. These data are combined with fluorescence (dye leakage) and quartz crystal microbalance studies to investigate the membrane interaction for these two wildtype peptides. It was found that the peptides penetrate the membrane in a trans-membrane manner. The amount of peptide uptake by the membrane is proportional to the concentration of the peptide, however, this appears to be a dynamic equilibrium which can be almost completely reversed by addition of buffer medium. Interestingly, a small residual mass remains within the membrane and the amount of peptide remaining in the membrane is a function of the buffer-salt concentration viz. in high salt, the residual peptide mass remaining is small whereas at low salt concentration, a larger mass of peptide remains bound. These results support a direct membrane penetration mechanism by the wild type apidaecins 1a and 1b. In both cases the peptide–membrane interaction has a negligible effect on the membrane, although, in high salt a permanent change in the membrane does occur at the highest peptide concentration which does not recover following peptide removal.

The model peptides glycylglycyltyrosylalanine (Gly-Gly-Tyr-Ala), glycylglycylthreonylalanine (Gly-Gly-Thr-Ala) and glycylglycylserylalanine (Gly-Gly-Ser-Ala) were phosphorylated at the hydroxyl groups of their tyrosyl, threonyl and seryl residues, respectively, and characterized by 31P and 1H NMR spectroscopy. The pKa-value of the phosphoryl group in the tyrosine-containing peptide determined from the pH dependence of chemical shifts is 5.9, the 31P chemical shifts at low pH (4.0) and high pH (8.0) are -3.8 and 0.2 ppm, respectively. Phosphorylation also leads to significant shifts of the 1H NMR resonances of the tyrosine residue; the amide resonance is shifted -0.02 ppm, the H alpha resonance 0.06 ppm, the H beta resonances 0.10 and -0.04 ppm, the H delta resonances 0.02 ppm and the H epsilon resonances 0.26 ppm. The pKa-value of the phosphoryl group in the threonine peptide determined from the pH dependence of chemical shifts is 6.1; the 31P chemical shifts at low pH (4.0) and high pH (8.0) are -0.1 and 4.8 ppm, respectively. The corresponding values for the serine peptide are 6.1 (pKa), 0.6 ppm and 4.9 ppm. Phosphorylation also leads to significant shifts of the 1H NMR resonances of the threonine and serine residues. In the threonine residue the amide resonance is shifted 0.25 ppm, the H alpha-resonance -0.43 ppm, the H beta-resonance 0.03 ppm and the H gamma-resonance 0.09 ppm. In the serine residue the amide resonance is shifted 0.21 ppm, the H alpha-resonance -0.17 ppm, and the H beta-resonances 0.17 ppm.

Developing new biopolymer-based materials with bio-identical properties is a significant challenge in modern science. One interesting route to this goal involves the biomineralization of collagen, a pre-structured and widely available protein, into a material with interesting properties. A prerequisite for biomineralization is the ability of cations (e.g., calcium) to bind to the protein and to result in concert with appropriate anions (e.g., phosphate) in composite material with e.g., bone-like properties. In order to increase the number of binding sites it is necessary to modify the protein prior to mineralization. For this glucuronic acid (GA) was used due to its carbonyl and carboxyl groups to derivatize proteinogenic amino groups transferring them into negatively charged carboxyl groups. Our experiments showed for the first time, that Nepsilon-carboxymethyllysine is the major product of in vitro non-enzymatic glycosylation of collagen by glucuronic acid. For an unequivocal determination of the reaction products, the lysine residues of collagen and of the model peptide were carboxymethylated through a reductive alkylation with glyoxalic acid and compared to the glucuronic acid derivatives. Beside their identical mass spectra the common structure elements could be confirmed with FTIR. Thus, in the context of matrix engineering, by producing Nepsilon-carboxymethyllysine, glucuronic acid offers a convenient way of introducing additional stable acidic groups into protein matrices.

A separation of mono, doubly and triply phosphorylated isomers was developed with CZE with an aqueous electrolyte containing 3.9 mol/L formic acid and 30% v/v trifluoroethanol. Thus a mixture of ten phosphopeptides corresponding to the human tau sequence 226-240 was separated within 70 min. Although peptides with different phosphorylation degrees, i.e. 0-3 phosphate groups, were well separated, some of the phosphopeptide isomers containing one or two phosphate groups were only partially separated. The electrolyte system is compatible with both MALDI- and ESI-MS, allowing a direct coupling, and thus could have some interesting applications in proteomics.

Protein and peptide therapeutics with good in vitro activities often fail due to poor bioavailability, circulation lifetime, and immunogenicity. PEGylation, i.e. conjugation of polyethylene glycol (PEG), significantly improves serum stability and renal clearance besides reducing the immunogenicity and thus enhances pharmacokinetics and tolerance in vivo. Several PEGylated drugs are marketed including several top-selling blockbusters. However, PEGylation can mask the binding site, especially in peptides, and thereby reduce the activity drastically, which is only rarely compensated by the improved bioavailability. Prodrug strategies using temporary PEGylation, i.e. the authentic drug is released from a PEG-linked precursor by hydrolysis or enzymatic degradation, can overcome these weaknesses. Recently, we reported a strategy coupling PEG via a peptide linker cleaved C-terminally by trypsin-like proteases in blood to release the unmasked therapeutic peptide. Here, we designed twelve short peptide linkers (four or five residues) to tune the release-rates of oncocin Onc112, a proline-rich antimicrobial peptide. In 25% aqueous mouse serum, Onc112 was released with half-life times from 0.5 to 12h. When elongated N-terminally with 5kDa ɑ-methoxy-ω-mercapto PEG as thioether, the half-life times of the prodrugs ranged from 7 to 42h in full mouse serum. Conjugation of a 20kDa instead of the 5kDa PEG increased the half-life times more than twofold, whereas longer peptide linkers up to twelve residues increased them only slightly. In all cases, Onc112 was released continuously providing stable peptide levels for at least 16h. The kinetics will allow the specific design of PEG-linker-drug-combinations for optimizing the pharmacokinetics of promising peptide therapeutics.

Differences in the protein distribution of various protein isolates from Lupinus angustifolius L. Vitabor were identified as affected by the isolation procedure (alkaline and/or salt-induced extraction followed by isoelectric and/or dilutive precipitation). Protein isolates extracted in alkaline solution showed higher protein yields (26.4-31.7%) compared to salt-induced extraction (19.8-30.0%) or combined alkaline and salt-induced extraction (23.3-25.6%). Chemical variations among the protein isolates especially occurred within the albumins. Protein isolates precipitated isoelectrically showed the highest contents, whereas protein isolates precipitated by dilutive showed the lowest contents of conglutin delta. Furthermore, the alkaline subunits of conglutin alpha and conglutin gamma decreased during alkaline extraction compared to salt-induced extraction. A decrease in protein-bound polar and basic amino acids was shown after protein isolation. In contrast, the amounts of nonpolar, aliphatic, aromatic, hydroxylated and sulfur-rich amino acids were higher in the lupin protein isolates compared to the lupin flakes. However, the functional side chains could not be related to the specific molecular arrangements of the protein isolates, as a similar amino acid composition was found among the protein isolates.

The detection and identification of O-phosphorylation sites in proteins with mass spectrometry remains a challenge. A common approach to analyse these modifications is to enrich phosphopeptides by immobilized metal affinity chromatography (IMAC) prior to mass spectrometric analysis. In this study two commercially available IMAC kits based on Fe(III)-ions immobilized on magnetic beads and Ga(III)-ions immobilized on a chelate-resin, have been investigated and the binding efficiency of peptide mixtures containing non-phosphorylated, singly, doubly and triply phosphorylated peptides have been tested.

By combining enzyme-linked immunosorbent assay (ELISA) and optical tweezers-assisted dynamic force spectroscopy (DFS), we identify for the first time the binding epitope of the phosphorylation-specific monoclonal antibody (mAb) HPT-101 to the Alzheimer's disease relevant peptide tau[pThr231/pSer235] on the level of single amino acids. In particular, seven tau isoforms are synthesized by replacing binding relevant amino acids by a neutral alanine (alanine scanning). From the binding between mAb HPT-101 and the alanine-scan derivatives, we extract specific binding parameters such as bond lifetime τ0, binding length x(ts), free energy of activation ΔG (DFS) and affinity constant K(a) (ELISA, DFS). Based on these quantities, we propose criteria to identify essential, secondary and non-essential amino acids, being representative of the antibody binding epitope. The obtained results are found to be in full accord for both experimental techniques. In order to elucidate the microscopic origin of the change in binding parameters, we perform molecular dynamics (MD) simulations of the free epitope in solution for both its parent and modified form. By taking the end-to-end distance d(E-E) and the distance between the α-carbons d(C-C) of the phosphorylated residues as gauging parameters, we measure how the structure of the epitope depends on the type of substitution. In particular, whereas d(C-C) is sometimes conserved between the parent and modified form, d(E-E) strongly changes depending on the type of substitution, correlating well with the experimental data. These results are highly significant, offering a detailed microscopic picture of molecular recognition.

Objectives To evaluate the efficacy of antimicrobial peptide Onc112 in a lethal Escherichia coli infection model and the pharmacokinetics of Onc72 and Onc112 administered intravenously or intraperitoneally in mice. Methods Onc72, Onc112 and their major metabolites in blood, kidneys, liver, brain and urine were quantified by MS using multiple reaction monitoring (MRM) and isotope-labelled peptides. Results Onc112 rescued all animals when administered intraperitoneally at a dose of 2.5 mg/kg and was thus slightly more efficient than Onc72. The MRM method provided limits of quantification in plasma, urine and kidney, liver and brain homogenates of 7-80 μg/L, well below the MICs of 2-4 mg/L. Onc72 and Onc112 reached all organs within 10 min when administered intraperitoneally (5 mg/kg). Their initial concentrations in plasma were 11.9 and 22.6 mg/L, respectively, with elimination t1/2 values of ∼14 and 21 min. The peptide concentrations in blood remained above their MICs for 20 min for Onc72 and 80 min for Onc112. The highest peptide concentrations were detected in kidney homogenates, which also contained the highest content of metabolites, indicating, together with the results from analysis of urine samples, that both peptides are cleared through the kidneys. Conclusions Onc72 and Onc112 reach organs, including the brain, within 10 min after intravenous and intraperitoneal administration. Onc112 remained in blood at concentrations above its MIC for 80 min. The pharmacokinetic profiles explain the high in vivo efficacies in models of systemic infection and indicate the potential use of these agents for the treatment of urinary tract infections.

The opportunistic Gram-negative bacterium Pseudomonas aeruginosa has a low susceptibility to common antibiotics. Additionally, around 15% of all clinical isolates bear acquired resistance genes. Thus, the development of new antibiotics to combat this pathogen in pneumonia, urinary tract infections, and bacteremia, represents an urgent task. The activity spectrum of the proline-rich antimicrobial peptide apidaecin 1b, originally isolated from honeybees (Apis mellifera), was extended in previous studies to further human pathogens including P. aeruginosa. However, the in vitro activity of the optimized peptide Api137 is limited to diluted medium conditions. Thus, we synthesized 323 analogs of Api137 on cellulose membranes using the SPOT strategy by substituting each residue individually by 19 other amino acids or deleting the residue. The peptides were deprotected with trifluoroacetic acid and cleaved with aqueous trimethylamine as C-terminal acids providing around 30 μg crude peptide per spot. This amount allowed determining the minimal inhibitory concentrations in a microdilution broth assay. The most promising substitutions were selected to synthesize 44 doubly and triply substituted Api137 analogs on the membrane. The 19 best peptides were synthesized at a larger scale and purified. Eight triply substituted Api137 analogs were up to 16-fold more active against P. aeruginosa at high medium concentrations without losing activities against Klebsiella pneumoniae and Acinetobacter baumannii and only slightly against Escherichia coli. The eight most active Api137 analogs were non-hemolytic to human erythrocytes and non-toxic to HeLa cells.

The study investigated the influence of the xanthophyll cycle pigments diadinoxanthin (DD) and diatoxanthin (Dt) on the spectroscopic characteristics, structure and protein composition of isolated fucoxanthin chlorophyll protein (FCP) complexes of the pennate diatom Phaeodactylum tricornutum. 77 K fluorescence emission spectra revealed that Dt-containing FCP complexes showed a characteristic long wavelength fluorescence emission at 700 nm at a pH-value of 5 whereas DD-enriched FCPs retained the typical 680 nm fluorescence emission maximum of isolated FCPs. The 700 nm emission in Dt-containing FCPs indicates an aggregation of antenna complexes and is a typical feature of the quenching site Q1 in recent models for non-photochemical fluorescence quenching (NPQ). A comparable long-wavelength fluorescence emission was found in FCP complexes prepared with either triton X-100 or n-dodecyl β-D-maltoside as detergent. A treatment of the FCP complexes at low pH-values in the presence of a high concentration of Mg2+ ions showed that the extent of FCP aggregation which leads to the 700 nm fluorescence emission is different from the macro-aggregation of antenna complexes in higher plants. Protein analyses by mass spectrometry showed that the protein composition of the DD- and Dt-enriched FCP complexes was comparable. However, the Lhcf6 and Lhcr1 polypeptides were only found in Dt-enriched FCPs isolated with dodecyl maltoside whereas the Lhcf17 protein was only detected in DD-enriched FCPs prepared with triton. With respect to low pH-induced antenna aggregation it is important that the Lhcx1 protein was found in both DD- and Dt-enriched FCPs, albeit with only two peptides with confident scores.