Adding Color to Mass Spectra of Biopolymers: Charge Determination Analysis (CHARDA) Assigns Charge State to Every Ion Peak.

Traditionally, mass spectrometry (MS) output is the ion abundance plotted versus the ionic mass-to-charge ratio m/z. While employing only commercially available equipment, Charge Determination Analysis (CHARDA) adds a third dimension to MS, estimating for individual peaks their charge states z starting from z = 1 and color coding z in m/z spectra. CHARDA combines the analysis of ion signal decay rates in the time-domain data (transients) in Fourier transform (FT) MS with the interrogation of mass defects (fractional mass) of biopolymers. Being applied to individual isotopic peaks in a complex protein tandem (MS/MS) data set, CHARDA aids peptide mass spectra interpretation by facilitating charge-state deconvolution of large ionic species in crowded regions, estimating z even in the absence of an isotopic distribution (e.g., for monoisotopic mass spectra). CHARDA is fast, robust, and consistent with conventional FTMS and FTMS/MS data acquisition procedures. An effective charge-state resolution Rz ≥ 6 is obtained with the potential for further improvements.

The Antioxidant and Proapoptotic Effects of Sternbergia clusiana Bulb Ethanolic Extract on Triple-Negative and Estrogen-Dependent Breast Cancer Cells In Vitro.

BACKGROUND: Sternbergia clusiana belongs to the Amaryllidaceae family and is recognized for the valuable biological activity of its major bioactive compounds. The aim of the current is to evaluate the anticancer effects of the ethanolic bulb extract of Sternbergia clusiana (ScBEE) on breast cancer cells in vitro and to further reveal the underlying cellular mechanism. METHODS: An MTS cell viability assay was performed on MDA-MB-231 and MCF-7 cells, along with cell cycle analysis, cell death ELISA, Western blot analysis and an ROS production assay to decipher the mechanism of death. LC-MS/MS was also performed to identify the chemical composition of this ethanolic extract. RESULTS: The results show a selective antiproliferative effect on both cell lines with no effect on normal mesenchymal stem cells. Further analysis suggested the activation of the apoptotic pathway as reflected by the increase in cellular and DNA fragmentation and alterations in apoptotic proteins such as Bax, Bcl-2 and c-PARP. ScBEE was also found to exhibit antioxidant effect, as shown by a decrease in ROS production. The underlying mechanism of action was explained by the presence of several bioactive compounds identified by LC-MS/MS, including alkaloids, terpenoids and phenols, which are elaborated in the manuscript. CONCLUSION: This study highlights the antioxidant and anticancerous properties of S.clusiana for breast cancer treatment.

Tyrosine bioconjugation with hypervalent iodine.

Hypervalent iodine reagents have recently emerged as powerful tools for late-stage peptide and protein functionalization. Herein we report a tyrosine bioconjugation methodology for the introduction of hypervalent iodine onto biomolecules under physiological conditions. Tyrosine residues were engaged in a selective addition onto the alkynyl bond of ethynylbenziodoxolones (EBX), resulting in stable vinylbenziodoxolones (VBX) bioconjugates. The methodology was successfully applied to peptides and proteins and tolerated all other nucleophilic residues, with the exception of cysteine. The generated VBX were further functionalized by palladium-catalyzed cross-coupling and azide-alkyne cycloaddition reactions. The method could be successfully used to modify bioactive natural products and native streptavidin to enable thiol-mediated cellular uptake.

Simultaneous mass spectrometry analysis of cisplatin with oligonucleotide-peptide mixtures: implications for the mechanism of action.

Although genomic DNA is the primary target of anticancer platinum-based drugs, interactions with proteins also play a significant role in their overall activity. In this study, competitive binding of cisplatin with an oligonucleotide and two peptides corresponding to segments of H2A and H2B histone proteins was investigated by mass spectrometry. Following the determination of the cisplatin binding sites on the oligonucleotide and peptides by tandem mass spectrometry, competitive binding was studied and transfer of platinum fragments from the platinated peptides to the oligonucleotide explored. In conjunction with previous studies on the nucleosome, the results suggest that all four of the abundant histone proteins serve as a platinum drug reservoir in the cell nucleus, providing an adduct pool that can be ultimately transferred to the DNA.

The pro-apoptotic properties of a phytonutrient rich infusion of A. cherimola leaf extract on AML cells.

Annonaceae family has broad uses in herbal medicine for treatment of several diseases, whether through seeds' or leaves' extracts. The present study investigates the antiproliferative and antitumor activity of Annona cherimola aqueous leaf (AAL) extract/infusion in acute myeloid leukemia (AML) cell lines in vitro. High-resolution LC-MS was first used to analyze the composition of the aqueous extract. Cell proliferation assay, Annexin V staining, cell cycle analysis, dual Annexin V/PI staining, cell death quantification by ELISA, ROS level detection and Western Blotting were then performed to elucidate the therapeutic effects of AAL extract. The results obtained revealed a potent antioxidant activity of AAL extract. Moreover, the extract exhibited dose- and time-dependent antiproliferative effects on AML cell lines by decreasing cell viability with an IC50 of 5.03% (v/v) at 24 h of treatment of KG-1 cells. This decrease in viability was accompanied with a significant increase in apoptotic cell death with cell cycle arrest and flipping of the phosphatidylserine from the inner to the outer leaflet of the cell membrane. The respective overexpression and downregulation of proapoptotic proteins like cleaved caspase-8, cleaved PARP-1 and Bax and antiapoptotic proteins like Bcl-2 further validated the apoptotic pathway induced by AAL on AML cells. Finally, LC-MS revealed the presence of several compounds like fatty acids, terpenes, phenolics, cinnamic acids and flavonoids that could contribute to the antioxidant and anti-cancer effects of this herbal infusion. In addition to the generally known nutritional effects of the Annona cherimola fruit and leaves, the presented data validates the antioxidant and anti-cancerous effects of the leaf infusion on AML cell lines, proposing its potential therapeutic use against acute myeloid leukemia with future in vivo and clinical trials.

A structure-based mechanism of cisplatin resistance mediated by glutathione transferase P1-1.

Cisplatin [cis-diamminedichloroplatinum(II) (cis-DDP)] is one of the most successful anticancer agents effective against a wide range of solid tumors. However, its use is restricted by side effects and/or by intrinsic or acquired drug resistance. Here, we probed the role of glutathione transferase (GST) P1-1, an antiapoptotic protein often overexpressed in drug-resistant tumors, as a cis-DDP-binding protein. Our results show that cis-DDP is not a substrate for the glutathione (GSH) transferase activity of GST P1-1. Instead, GST P1-1 sequesters and inactivates cisplatin with the aid of 2 solvent-accessible cysteines, resulting in protein subunits cross-linking, while maintaining its GSH-conjugation activity. Furthermore, it is well known that GST P1-1 binding to the c-Jun N-terminal kinase (JNK) inhibits JNK phosphorylation, which is required for downstream apoptosis signaling. Thus, in turn, GST P1-1 overexpression and Pt-induced subunit cross-linking could modulate JNK apoptotic signaling, further confirming the role of GST P1-1 as an antiapoptotic protein.

Biochemical and biophysical characterization of ruthenation of BRCA1 RING protein by RAPTA complexes and its E3 ubiquitin ligase activity.

RAPTA compounds, ([Ru(η6-arene)(PTA)Cl2], PTA = 1,3,5-triaza-7-phosphaadamantane), have been reported to overcome drug resistance in cisplatin resistant cells. However, the exact mechanism of these complexes is still largely unexplored. In this study, the interaction of some RAPTA compounds with the N-terminal fragment of the BRCA1 RING domain protein was investigated. The binding of the RAPTA compounds to the BRCA1 protein resulted in a release of Zn2+ ions in a dose and time dependent manner, as well as thermal alteration of ruthenated-BRCA1 proteins. Electron Transfer Dissociation (ETD) fragmentation mass spectrometry revealed the preferential binding sites of the RAPTA complexes on the BRCA1 zinc finger RING domain at a similar short peptide stretch, Cys24Lys25Phe26Cys27Met28Leu29 and Lys35 (residues 44-49 and 55 on full length BRCA1). Changes in the conformation and binding constants of ruthenium-BRCA1 adducts were established, resulting in inactivation of the RING heterodimer BRCA1/BARD1-mediated E3 ubiquitin ligase function. These findings could provide mechanistic insight into the mode of action of RAPTA complexes for on tested BRCA1 model protein.

Structure and Biophysical Properties of a Triple-Stranded Beta-Helix Comprising the Central Spike of Bacteriophage T4.

Gene product 5 (gp5) of bacteriophage T4 is a spike-shaped protein that functions to disrupt the membrane of the target cell during phage infection. Its C-terminal domain is a long and slender ß-helix that is formed by three polypeptide chains wrapped around a common symmetry axis akin to three interdigitated corkscrews. The folding and biophysical properties of such triple-stranded ß-helices, which are topologically related to amyloid fibers, represent an unsolved biophysical problem. Here, we report structural and biophysical characterization of T4 gp5 ß-helix and its truncated mutants of different lengths. A soluble fragment that forms a dimer of trimers and that could comprise a minimal self-folding unit has been identified. Surprisingly, the hydrophobic core of the ß-helix is small. It is located near the C-terminal end of the ß-helix and contains a centrally positioned and hydrated magnesium ion. A large part of the ß-helix interior comprises a large elongated cavity that binds palmitic, stearic, and oleic acids in an extended conformation suggesting that these molecules might participate in the folding of the complete ß-helix.

Chemical macrocyclization of peptides fused to antibody Fc fragments.

To extend the plasma half-life of a bicyclic peptide antagonist, we chose to link it to the Fc fragment of the long-lived serum protein IgG1. Instead of chemically conjugating the entire bicyclic peptide, we recombinantly expressed its peptide moiety as a fusion protein to an Fc fragment and subsequently cyclized the peptide by chemically reacting its three cysteine residues with tris-(bromomethyl)benzene. This reaction was efficient and selective, yielding completely modified peptide fusion protein and no side products. After optimization of the linker and the Fc fragment format, the bicyclic peptide was fully functional as an inhibitor (K(i) = 76 nM) and showed an extended terminal half-life of 1.5 days in mice. The unexpectedly clean reaction makes chemical macrocyclization of peptide-Fc fusion proteins an attractive synthetic approach. Its good compatibility with the Fc fragment may lend the bromomethylbenzene-based chemistry also for the generation of antibody-drug conjugates.

UV radiation induces genome-mediated, site-specific cleavage in viral proteins.

Much research has been dedicated to understanding the molecular basis of UV damage to biomolecules, yet many questions remain regarding the specific pathways involved. Here we describe a genome-mediated mechanism that causes site-specific virus protein cleavage upon UV irradiation. Bacteriophage MS2 was disinfected with 254 nm UV, and protein damage was characterized with ESI- and MALDI-based FT-ICR, Orbitrap, and TOF mass spectroscopy. Top-down mass spectrometry of the products identified the backbone cleavage site as Cys46-Ser47 in the virus capsid protein, a location of viral genome-protein interaction. The presence of viral RNA was essential to inducing backbone cleavage. The similar bacteriophage GA did not exhibit site-specific protein cleavage. Based on the major protein fragments identified by accurate mass analysis, a cleavage mechanism is proposed by radical formation. The mechanism involves initial oxidation of the Cys46 side chain followed by hydrogen atom abstraction from Ser47 C(α). Computational protein QM/MM studies confirmed the initial steps of the radical mechanism. Collectively, this study describes a rare incidence of genome-induced protein cleavage without the addition of sensitizers.

Efficient orthogonal bioconjugation of dendrimers for synthesis of bioactive nanoparticles.

Nanoparticles carrying biologically active functional sets (e.g., targeting moiety, payload, tracer) have potential use in a wide range of clinical applications. Though complex, such constructions should, as far as possible, have a defined molecular architecture and be monodisperse. However, the existing methods to achieve this goal are unsuitable for the incorporation of peptides and proteins, and those that provide for orthogonal introduction of two different types of functional element are incompatible with the use of commercially available materials. In this study, we have developed approaches for the production of nanoparticles based on commercially available polyamidoamine (PAMAM) dendrimers. First, we identified an optimized oxime conjugation strategy under which complex dendrimers can be fully decorated not only with model peptides, but also with recombinant proteins (insulin was taken as an example). Second, we developed a strategy based on a two-chain covalent heterodendrimer (a "diblock") based on cystamine core PAMAM dendrimers and used it to generate heterodendrimers, into which a peptide array and a mannose array were orthogonally introduced. Finally, by incorporating a functionalized linker into the diblock architecture we were able to site-specifically introduce a third functional element into the nanoparticle. We exemplified this approach using fluorescein, a mannose array, and a peptide array as the three functionalities. We showed that incorporation of a mannose array into a nanoparticle strongly and specifically enhances uptake by sentinel cells of the immune system, an important property for vaccine delivery applications. These PAMAM dendrimer-based approaches represent a robust and versatile platform for the development of bioactive nanoparticles.

Platelet-active substances in the venom of Bothrops moojeni snake-a novel evaluation method using whole blood aggregometry.

The objective of the present study was an investigation of the crude Bothrops moojeni venom, aiming at the identification of new compounds with platelet-activating or -inhibiting activity. The venom was separated by gel filtration chromatography into 18 fractions, which were tested by means of whole blood aggregometry for their activities affecting the aggregation of blood platelets. In order to eliminate interferences caused by prothrombin activators or thrombin like-enzymes, which are frequently present in snake venoms, a test method for screening protein mixtures was developed. To avoid clotting of the blood samples, the thrombin inhibitor hirudin and the synthetic inhibitor of fibrin polymerization Pefabloc FG were applied. In the present study, a platelet aggregation activator with an activity resembling thrombocytin from B. atrox was identified in one of the examined venom fractions. In addition, a platelet antagonist-most likely a disintegrin-with broad inhibitory activity against aggregation triggered by collagen, adenosine diphosphate and thrombin receptor activating peptide, was identified.

A post-modification approach to peptide foldamers.

Hydrophilic/hydrophobic patterning is a powerful strategy to control folding in non-natural polymers/oligomers. In this contribution, we present a novel strategy for the preparation of alternating hydrophilic/hydrophobic patterned non-natural peptide foldamers. This strategy relies on the post-modification of a reactive peptide precursor that can be prepared via standard solid phase peptide synthesis without the need for side chain protective groups. The peptide scaffolds consisted of an alternating sequence of l-leucine and l-allylglycine residues. Using thiol-ene chemistry, the double bonds in the side chains of the l-allylglycine units could be post-modified with cysteamine hydrochloride, thioglycolic acid, 1-thioglycerol or 2,3,4,6-tetra-O-acetyl-thio-beta-d-glucopyranose to afford alternating hydrophilic/hydrophobic patterned peptides. In agreement with the alternating hydrophilic/hydrophobic patterned primary structure, cysteamine and thioglycolic acid post-modified octapeptides were found to adopt a beta-sheet secondary structure in basic or acidic aqueous media, respectively. The proposed synthetic approach is not only of interest to generate diverse libraries of peptide foldamers from a limited number of reactive precursor scaffolds, but may also represent an attractive, general strategy for the synthesis of peptides with complex side chain functionalities that are not easily accessible via standard solid phase techniques.

High throughput screening of bradykinin-potentiating peptides in Bothrops moojeni snake venom using precursor ion mass spectrometry.

Snake venoms are known to be an extensive source of bioactive peptides. Bradykinin-potentiating peptides (BPPs) are inhibitors of the angiotensin-converting enzyme that have already been identified in the venom of many snake, scorpion, spider and batrachian species. Their most characteristic structural features are an invariable N-terminal pyroglutamate residue (pGlu or Z) and two consecutive proline residues at the C-terminus. Fragmentation of BPPs by collision-induced dissociation during electrospray tandem mass spectrometry analysis (ESI-MS/MS) generates a predominant signal at m/z 213.1 corresponding to the y-ion of the terminal Pro-Pro fragment. In addition, signals at m/z 226.1 and 240.1 that correspond to the b ions of the N-terminus pGlu-Asn and pGlu-Lys, respectively, can often be observed. Based on these structural determinants, the present work describes an original methodology for the discovery of BPPs in natural extracts using liquid chromatography coupled to ESI-MS/MS operated in precursor ion-scan mode. The venom of the Bothrops moojeni snake was used as a model and the methodology was applied for subsequent structural analysis of the identified precursors by tandem mass spectrometry on quadrupole-time-of-flight (Q-TOF) and matrix-assisted laser-desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) instruments. More than 40 peptides below 2500 Da could be detected, among them 20 were shown to belong to the BPP-like family including the related tripeptides pGlu-Lys-Trp and pGlu-Asn-Trp. A total of 15 new sequences have been identified using this approach.

The venom of the snake genus Atheris contains a new class of peptides with clusters of histidine and glycine residues.

We investigated venoms from members of the genus Atheris (Serpentes, Viperidae), namely the rough scale bush viper (Atheris squamigera), the green bush viper (A. chlorechis) and the great lakes bush viper (A. nitschei), using mass spectrometry-based strategies, relying on matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) with de novo peptide sequencing. We discovered a set of novel peptides with masses in the 2-3 kDa range and containing poly-His and poly-Gly segments (pHpG). Complete primary structural elucidation and confirmation of two sequences by Edman degradation indicated the consensus sequence EDDH(9)GVG(10). Bioinformatic investigations in protein sequence databanks did not show relevant homology with known peptides or proteins. However, a more extensive investigation of data in nucleic acid databases revealed some similarities to the precursor sequences of bradykinin potentiating peptides (BPP) and C-type natriuretic peptides (CNP), agents that are known to affect the cardiovascular system by acting on specific metalloproteases and receptors. The novel pHpG peptides found in Atheris venoms might also act on the cardiovascular system by inhibiting particular metalloproteases, which however remain to be identified.

Effective depletion of albumin using a new peptide-based affinity medium.

Blood plasma and serum are very useful samples for the detection, identification and quantitation of proteins associated with both health and disease. However, analysis of plasma and serum is a challenge because traces of interesting polypeptides and proteins can be dominated by the very high concentration of albumin present. Albumin may be depleted by adsorption to immunoaffinity columns or to columns containing dyes such as Cibacron Blue, or by ultrafiltration, but these methods are far from ideal. We describe a new peptide-based affinity medium which is effective for removing albumin and is very specific. The albumin-binding capacity is at least 14 mg per mL of gel. The material may be reused hundreds of times after a simple regeneration step involving NaOH, with full retention of specificity and capacity. The material was tested with human and monkey plasma and serum and rat serum, and has been used to deplete litre volumes of human plasma. The development of other peptide-based affinity media to deplete abundant proteins is briefly discussed.

Anti-microbial peptides: from invertebrates to vertebrates.

Gene-encoded anti-microbial peptides (AMPs) are widespread in nature, as they are synthesized by microorganisms as well as by multicellular organisms from both the vegetal and the animal kingdoms. These naturally occurring AMPs form a first line of host defense against pathogens and are involved in innate immunity. Depending on their tissue distribution, AMPs ensure either a systemic or a local protection of the organism against environmental pathogens. They are classified into three major groups: (i) peptides with an alpha-helical conformation (insect cecropins, magainins, etc.), (ii) cyclic and open-ended cyclic peptides with pairs of cysteine residues (defensins, protegrin, etc.), and (iii) peptides with an over-representation of some amino acids (proline rich, histidine rich, etc.). Most AMPs display hydrophobic and cationic properties, have a molecular mass below 25-30 kDa, and adopt an amphipathic structure (alpha-helix, beta-hairpin-like beta-sheet, beta-sheet, or alpha-helix/beta-sheet mixed structures) that is believed to be essential to their anti-microbial action. Interestingly, in recent years, a series of novel AMPs have been discovered as processed forms of large proteins. Despite the extreme diversity in their primary and secondary structures, all natural AMPs have the in vitro particularity to affect a large number of microorganisms (bacteria, fungi, yeast, virus, etc.) with identical or complementary activity spectra. This review focuses on AMPs forming alpha-helices, beta-hairpin-like beta-sheets, beta-sheets, or alpha-helix/beta-sheet mixed structures from invertebrate and vertebrate origins. These molecules show some promise for therapeutic use.

Industrial-scale proteomics: from liters of plasma to chemically synthesized proteins.

Human blood plasma is a useful source of proteins associated with both health and disease. Analysis of human blood plasma is a challenge due to the large number of peptides and proteins present and the very wide range of concentrations. In order to identify as many proteins as possible for subsequent comparative studies, we developed an industrial-scale (2.5 liter) approach involving sample pooling for the analysis of smaller proteins (M(r) generally < ca. 40 000 and some fragments of very large proteins). Plasma from healthy males was depleted of abundant proteins (albumin and IgG), then smaller proteins and polypeptides were separated into 12 960 fractions by chromatographic techniques. Analysis of proteins and polypeptides was performed by mass spectrometry prior to and after enzymatic digestion. Thousands of peptide identifications were made, permitting the identification of 502 different proteins and polypeptides from a single pool, 405 of which are listed here. The numbers refer to chromatographically separable polypeptide entities present prior to digestion. Combining results from studies with other plasma pools we have identified over 700 different proteins and polypeptides in plasma. Relatively low abundance proteins such as leptin and ghrelin and peptides such as bradykinin, all invisible to two-dimensional gel technology, were clearly identified. Proteins of interest were synthesized by chemical methods for bioassays. We believe that this is the first time that the small proteins in human blood plasma have been separated and analyzed so extensively.

Lead optimization of antifungal peptides with 3D NMR structures analysis.

Antimicrobial peptides are key components of the innate immune response in most multicellular organisms. These molecules are considered as one of the most innovative class of anti-infective agents that have been discovered over the last two decades, and therefore, as a source of inspiration for novel drug design. Insect cystine-rich antimicrobial peptides with the CS alpha beta scaffold (an alpha-helix linked to a beta-sheet by two disulfide bridges) represent particularly attractive templates for the development of systemic agents owing to their remarkable resistance to protease degradation. We have selected heliomicin, a broad spectrum antifungal CS alpha beta peptide from Lepidoptera as the starting point of a lead optimization program based on phylogenic exploration and fine tuned mutagenesis. We report here the characterization, biological activity, and 3D structure of heliomicin improved analogs, namely the peptides ARD1, ETD-135, and ETD-151. The ARD1 peptide was initially purified from the immune hemolymph of the caterpillars of Archeoprepona demophoon. Although it differs from heliomicin by only two residues, it was found to be more active against the human pathogens Aspergillus fumigatus and Candida albicans. The peptides ETD-135 and ETD-151 were engineered by site-directed mutagenesis of ARD1 in either cationic or hydrophobic regions. ETD-135 and ETD-151 demonstrated an improved antifungal activity over the native peptides, heliomicin and ARD1. A comparative analysis of the 3D structure of the four molecules highlighted the direct impact of the modification of the amphipathic properties on the molecule potency. In addition, it allowed to characterize an optimal organization of cationic and hydrophobic regions to achieve best antifungal activity.