Photoaffinity labeling coupled to MS to identify peptide biological partners: Secondary reactions, for better or for worse?

Affinity photolabeling is a smart method to study noncovalent and transient interactions and provide a submolecular picture of the contacts between interacting partners. In this review, we will focus on the identification of peptide partners using photoaffinity labeling coupled to mass spectrometry in different contexts such as in vitro with a purified potential partner, in model systems such as model membranes, and with live cells using both targeted and nontargeted proteomics studies. Different biological partners will be described, among which glycoconjugates, oligonucleotides, peptides, proteins, and lipids, with the photoreactive label inserted either on the peptide of interest or on the potential partner. Particular attention will be paid to the observation and characterization of specific rearrangements following the photolabeling reaction, which can help characterize photoadducts and provide a better understanding of the interacting systems and environment.

α-Silylated Diazoalkynes: New Tools for Bioconjugation of Proteins.

α-Silylated diazoalkynes are stabilized diazo compounds that can selectively react with carboxylic residues in buffered aqueous media. In-situ fluoride induced desilylation increases this reactivity, leading to a very fast reaction. Application to the selective functionalization of RNase A, followed by post-functionalization using click chemistry, is described. These new reagents expand the toolbox for native protein modification at carboxylic residues.

Characterization of a New Immunosuppressive and Antimicrobial Peptide, DRS-DA2, Isolated from the Mexican Frog, Pachymedusa dacnicolor.

Inflammatory and antimicrobial diseases constitute a major burden for society, and fighting them is a WHO strategic priority. Most of the treatments available to fight inflammatory diseases are anti-inflammatory drugs, such as corticosteroids or immunomodulators that lack cellular specificity and lead to numerous side effects. In addition to suppressing undesired inflammation and reducing disease progression, these drugs lessen the immune system protective functions. Furthermore, treating infectious diseases is more and more challenging due to the rise of microbial resistance to antimicrobial drugs. Thus, controlling the inflammatory process locally without compromising the ability to combat infections is an essential feature in the treatment of inflammatory diseases. We isolated three forms (DRS-DA2N, DRS-DA2NE, and DRS-DA2NEQ) of the same peptide, DRS-DA2, which belongs to the dermaseptin family, from the Mexican tree frog Pachymedusa dacnicolor. Interestingly, DRS-DA2N and DRS-DA2NEQ exhibit a dual activity by inducing the death of leukocytes as well as that of Gram-negative and Gram-positive bacteria, including multiresistant strains, without affecting other cells such as epithelial cells or erythrocytes. We showed that the death of both immune cells and bacteria is induced rapidly by DRS-DA2 and that the membrane is permeabilized, leading to the loss of membrane integrity. We also validated the capacity of DRS-DA2 to regulate the pool of inflammatory cells in vivo in a mouse model of noninfectious peritonitis. After the induction of peritonitis, a local injection of DRS-DA2N could decrease the number of inflammatory cells locally in the peritoneal cavity without inducing a systemic effect, as no changes in the number of inflammatory cells could be detected in blood or in the bone marrow. Collectively, these data suggest that this peptide could be a promising tool in the treatment of inflammatory diseases, such as inflammatory skin diseases, as it could reduce the number of inflammatory cells locally without suppressing the ability to combat infections.

Analytical Tools for Dynamic Combinatorial Libraries of Cyclic Peptides.

Target-directed dynamic combinatorial chemistry is a very attractive strategy for the discovery of bioactive peptides. However, its application has not yet been demonstrated, presumably due to analytical challenges that arise from the diversity of a peptide library with combinatorial side-chains. We previously reported an efficient method to generate, under biocompatible conditions, large dynamic libraries of cyclic peptides grafted with amino acid's side-chains, by thiol-to-thioester exchanges. In this work, we present analytical tools to easily characterize such libraries by HPLC and mass spectrometry, and in particular to simplify the isomers' distinction requiring sequencing by MS/MS fragmentations. After structural optimization, the cyclic scaffold exhibits a UV-tag, absorbing at 415 nm, and an ornithine residue which favors the regioselective ring-opening and simultaneous MS/MS fragmentation, in the gas-phase.

Structural Bases for the Involvement of Phosphatidylinositol-4,5-bisphosphate in the Internalization of the Cell-Penetrating Peptide Penetratin.

Cell-penetrating peptides cross cell membranes through various parallel internalization pathways. Herein, we analyze the role of the negatively charged lipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the internalization of Penetratin. Contributions of both inner leaflet and outer leaflet pools of PI(4,5)P2 were revealed by quantifying the internalization of Penetratin in cells treated with PI(4,5)P2 binders. Studies on model systems showed that Penetratin has a strong affinity for PI(4,5)P2 and interacts selectively with this lipid, even in the presence of other negatively charged lipids, as demonstrated by affinity photo-crosslinking experiments. Differential scanning calorimetry experiments showed that Penetratin induces lateral segregation in PI(4,5)P2-containing liposomes, which was confirmed by coarse-grained molecular dynamics simulations. NMR experiments indicated that Penetratin adopts a stabilized helical conformation in the presence of PI(4,5)P2-containing membranes, with an orientation parallel to the bilayer plane, which was also confirmed by all-atom simulations. NMR and photo-crosslinking experiments also suggest a rather shallow insertion of the peptide in the membrane. Put together, our findings suggest that PI(4,5)P2 is a privileged interaction partner for Penetratin and that it plays an important role in Penetratin internalization.

Dynamic Amino Acid Side-Chains Grafting on Folded Peptide Backbone.

An efficient strategy for the synthesis of large libraries of conformationally defined peptides is reported, using dynamic combinatorial chemistry as a tool to graft amino acid side chains on a well-ordered 3D (3-dimension) peptide backbone. Combining rationally designed scaffolds with combinatorial side chains selection represents an alternative method to access peptide libraries for structures that are not genetically encodable. This method would allow a breakthrough for the discovery of protein mimetic for unconventional targets for which little is known.

Lipophilic quinolone derivatives: Synthesis and in vitro antibacterial evaluation.

This paper reports on the design of a series of 10 novel lipophilic piperazinyl derivatives of the 1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, their synthesis, their characterisation by 1H, 13C and 19F NMR, IR spectroscopy and HRMS, as well as their biological activity against bacteria of medical interest. Among these derivatives, 2 were as potent as the parent quinolone against Neisseriagonorrhoeae whereas all the compounds displayed lower activity than the parent quinolone against other bacteria of medical interest. Our results showing that the increased lipophilicity was deleterious for antibacterial activity may help to design new quinolone derivatives in the future, especially lipophilic quinolones which have been poorly investigated previously.

Photolabeling Strategies to Study Membranotropic Peptides Interacting with Lipids and Proteins in Membranes.

Membranotropic peptides is a class of peptides that exert their biological action at the level of cell membranes. Understanding how they interact with their different membrane binding partners (lipids, proteins, and/or glycoconjugates) is important to decipher their mechanism of action. Affinity photolabeling is a powerful method to study noncovalent interactions and provide a submolecular picture of the contacts between two interacting partners. In this review, we give a panorama of photolabeling-based studies of the interactions between membranotropic peptides and membranes using either photoreactive lipids or peptides.

Binding and crossing: Methods for the characterization of membrane-active peptides interactions with membranes at the molecular level.

Antimicrobial and cell-penetrating peptides have been the object of extensive studies for more than 60 years. Initially these two families were studied separately, and more recently parallels have been drawn. These studies have given rise to numerous methodological developments both in terms of observation techniques and membrane models. This review presents some of the most recent original and innovative developments in this field, namely droplet interface bilayers (DIBs), new fluorescence approaches, force measurements, and photolabelling.

Benzophenone Photoreactivity in a Lipid Bilayer To Probe Peptide/Membrane Interactions: Simple System, Complex Information.

Affinity photo-cross-linking coupled to mass spectrometry, using benzophenone (Bzp)-functionalized peptides, was used to study the noncovalent interactions of cell-penetrating peptides and lipid membranes. Using biomimetic lipid vesicles composed of saturated and unsaturated negatively charged lipids, DMPG (14:0), DPPG (16:0), DOPG (18:1 cis Δ9), 18:1 (trans Δ9) PG, and DLoPG (18:2 cis Δ9, 12), allowed observation of all the classical and less common reactivities of Bzp described in the literature by direct MS analysis: C═C double bond formation on saturated fatty acids, covalent adducts formation via classical C-C bond, and Paternò-Büchi oxetane formation followed or not by fragmentation (retro-Paternò-Büchi) as well as photosensitization of unsaturated lipids leading to lipid dimers. All these reactions can occur concomitantly in a single complex biological system: a membrane-active peptide inserted within a phospholipid bilayer. We also detect oxidation species due to the presence of radical oxygen species. This work represents a noteworthy improvement for the characterization of interacting partners using Bzp photo-cross-linking, and it shows how to exploit in an original way the different reactivities of Bzp in the context of a lipid membrane. We propose an analytical workflow for the interpretation of MS spectra, giving access to information on the CPP/lipid interaction at a molecular level such as depth of insertion or membrane fluidity in the CPP vicinity. An application of this workflow illustrates the role of cholesterol in the CPP/lipids interaction.

Peptidoglycan potentiates the membrane disrupting effect of the carboxyamidated form of DMS-DA6, a Gram-positive selective antimicrobial peptide isolated from Pachymedusa dacnicolor skin.

The occurrence of nosocomial infections has been on the rise for the past twenty years. Notably, infections caused by the Gram-positive bacteria Staphylococcus aureus represent a major clinical problem, as an increase in antibiotic multi-resistant strains has accompanied this rise. There is thus a crucial need to find and characterize new antibiotics against Gram-positive bacteria, and against antibiotic-resistant strains in general. We identified a new dermaseptin, DMS-DA6, produced by the skin of the Mexican frog Pachymedusa dacnicolor, with specific antibacterial activity against Gram-positive bacteria. This peptide is particularly effective against two multiple drug-resistant strains Enterococcus faecium BM4147 and Staphylococcus aureus DAR5829, and has no hemolytic activity. DMS-DA6 is naturally produced with the C-terminal carboxyl group in either the free or amide forms. By using Gram-positive model membranes and different experimental approaches, we showed that both forms of the peptide adopt an α-helical fold and have the same ability to insert into, and to disorganize a membrane composed of anionic lipids. However, the bactericidal capacity of DMS-DA6-NH2 was consistently more potent than that of DMS-DA6-OH. Remarkably, rather than resulting from the interaction with the negatively charged lipids of the membrane, or from a more stable conformation towards proteolysis, the increased capacity to permeabilize the membrane of Gram-positive bacteria of the carboxyamidated form of DMS-DA6 was found to result from its enhanced ability to interact with peptidoglycan.

Photoactivatable oligonucleotide probes to trap single-stranded DNA binding proteins: Updating the potential of 4-thiothymidine from a comparative study.

Photoaffinity labeling (PAL) in combination with recent developments in mass spectrometry is a powerful tool for studying nucleic acid-protein interactions, enabling crosslinking of both partners through covalent bond formation. Such a strategy requires a preliminary study of the most judicious photoreactive group to crosslink efficiently with the target protein. In this study, we report a survey of three different photoreactive nucleobases (including a guanine functionalized with a benzophenone or a diazirine and the zero-length agent 4-thiothymine) incorporated in 30-mer oligonucleotides (ODN) containing a biotin moiety for selective trapping and enrichment of single-stranded DNA binding proteins (SSB). First, the conditions and efficiency of the photochemical reaction with a purified protein using human replication protein A as the relevant model was studied. Secondly, the ability of the probe as bait to photocrosslink and enrich SSB in cell lysate was addressed. Among the different ODN probes studied, we showed that 4-thiothymine was the most relevant: i) it allows efficient and specific trapping of SSB in whole cell extracts in a similar extent as the widely used diazirine, ii) it features the advantages of a zero-length agent thus retaining the physicochemical properties of the ODN bait; iii) ODN including this photochemical agent are easily accessible. In combination with mass spectrometry, the probes incorporating this nucleobase are powerful tools for PAL strategies and can be added in the toolbox of the traditional photocrosslinkers for studying DNA-protein interactions.

Exploiting Benzophenone Photoreactivity To Probe the Phospholipid Environment and Insertion Depth of the Cell-Penetrating Peptide Penetratin in Model Membranes.

Penetratin (RQIKIWFQNRRMKWKK) enters cells by different mechanisms, including membrane translocation, thus implying that the peptide interacts with the lipid bilayer. Penetratin also crosses the membrane of artificial vesicles, depending on their phospholipid content. To evaluate the phospholipid preference of penetratin, as the first step of translocation, we exploited the benzophenone triplet kinetics of hydrogen abstraction, which is slower for secondary than for allylic hydrogen atoms. By using multilamellar vesicles of varying phospholipid content, we identified and characterized the cross-linked products by MALDI-TOF mass spectrometry. Penetratin showed a preference for negatively charged (vs. zwitterionic) polar heads, and for unsaturated (vs. saturated) and short (vs. long) saturated phospholipids. Our study highlights the potential of using benzophenone to probe the environment and insertion depth of membranotropic peptides in membranes.

A direct label-free MALDI-TOF mass spectrometry based assay for the characterization of inhibitors of protein lysine methyltransferases.

Histone lysine methylation is associated with essential biological functions like transcription activation or repression, depending on the position and the degree of methylation. This post-translational modification is introduced by protein lysine methyltransferases (KMTs) which catalyze the transfer of one to three methyl groups from the methyl donor S-adenosyl-L-methionine (AdoMet) to the amino group on the side chain of lysines. The regulation of protein lysine methylation plays a primary role not only in the basic functioning of normal cells but also in various pathologies and KMT deregulation is associated with diseases including cancer. These enzymes are therefore attractive targets for the development of new antitumor agents, and there is still a need for direct methodology to screen, identify, and characterize KMT inhibitors. We report here a simple and robust in vitro assay to quantify the enzymatic methylation of KMT by MALDI-TOF mass spectrometry. Following this protocol, we can monitor the methylation events over time on a peptide substrate. We detect in the same spectrum the modified and unmodified substrates, and the ratios of both signals are used to quantify the amount of methylated substrate. We first demonstrated the validity of the assay by determining inhibition parameters of two known inhibitors of the KMT SET7/9 ((R)-PFI-2 and sinefungin). Next, based on structural comparison with these inhibitors, we selected 42 compounds from a chemical library. We applied the MALDI-TOF assay to screen their activity as inhibitors of the KMT SET7/9. This study allowed us to determine inhibition constants as well as kinetic parameters of a series of SET7/9 inhibitors and to initiate a structure activity discussion with this family of compounds. This assay is versatile and can be easily adapted to other KMT substrates and enzymes as well as automatized.

Proteomic comparison of the EWS-FLI1 expressing cells EF with NIH-3T3 and actin remodeling effect of (R/W)9 cell-penetrating peptide.

EWS-FLI1 expression in NIH-3T3 fibroblasts has a profound impact on the phenotype, resulting in the cytoskeleton and adhesive capacity disorganization (EF cells). Besides this, (R/W)9, a cell-penetrating peptide (CPP), has an intrinsic actin remodeling activity in EF cells. To evaluate the impact of the oncogenic protein EWS-FLI1 on proteins expression levels, a quantitative comparison of tumoral EF and non-tumoral 3T3 proteomes was performed. Then to see if we could link the EWS-FLI1 oncogenic transformation to the phenotype reversion induced by (R/W)9, (R/W)9 influence on EF cells proteome was assessed. To our knowledge no such â¿¿CPPomicâ¿¿ study has been performed before. BIOLOGICAL SIGNIFICANCE: Up to now very few global quantitative proteomic studies have been published to help understand the oncogenic transformation induced by EWS-FLI1 fusion protein and leading to Ewing sarcoma development and dissemination. The comparison we did in this study between a model tumoral cell line EF and its non-tumoral counterpart (3T3) allowed us to highlight several features either common to most tumor types or specific to Ewing sarcoma. Particularly, lack of actin cytoskeleton organization could very likely be explained by the down-regulation of many important actin binding proteins. These results are in accordance with the hypothesis of a passive/stochastic mode of dissemination conferring Ewing sarcoma tumoral cell a high metastatic potential.

Photocross-Linked Peptide-Protein Complexes Analysis: A Comparative Study of CID and ETD Fragmentation Modes.

Protein-protein interactions are among the keys to organizing cellular processes in space and time. One of the only direct ways to identify such interactions in their cellular environment is to covalently bond the interacting partners to fix the interaction. Photocross-linking in living cells is thus a very promising technique. The feasibility of in cellulo photocross-linking reactions has been shown and mass spectrometry is a tool of choice to analyze photocross-linked proteins. However, the interpretation of the MS and MS/MS spectra of photocross-linked peptides remains one of the most important bottlenecks of the method and still limits its potential for large-scale applications (interactomics). Fundamental studies are still necessary to understand and characterize the fragmentation behavior of photocross-linked peptides. Here, we report the successful identification of the interaction sites in a well-characterized model of in vitro interaction between a protein and a peptide. We describe in detail the fragmentation pattern of these photocross-linked species in order to identify trends that could be generalized. In particular, we compare CID and ETD fragmentation modes (and HCD in a lesser extent), demonstrating the complementarity of both methods and the advantage of ETD for the analysis of photocross-linked species. The information should help further development of dedicated software to properly score MS/MS spectra of photocross-linked species.

LG3 fragment of endorepellin is a possible biomarker of severity in IgA nephropathy.

IgA nephropathy (IgAN), the most common primary glomerulonephritis, is characterized by deposition of IgA in the glomerular mesangium. The diagnosis of IgAN still requires a kidney biopsy that cannot easily be repeated in the same patient during follow-up. Therefore, identification of noninvasive urinary biomarkers would be very useful for monitoring patients with IgAN. We first used bidimensional electrophoresis (2DE) coupled to MALDI-TOF-TOF and Western blot to identify some urinary biomarkers associated with IgAN. Urine of IgAN patients showed an increase of albumin fragments, α-1-antitrypsin and α-1-ß-glycoprotein, along with a decrease of a single spot that was identified as the laminin G-like 3 (LG3) fragment of endorepellin. The urinary proteomes of 43 IgAN patients were compared to those of 30 healthy individuals by ELISA. Quantification of LG3 confirmed a significant decrease in the urine of IgAN patients compared to healthy controls, except in ten patients in whom LG3 was increased. These ten patients had a more severe disease with lower glomerular filtration rate values. We found a significant inverse correlation between LG3 levels and glomerular filtration rate in the 43 patients with IgAN, which was not observed in 65 patients with other glomerular diseases including membranous nephropathy (23), lupus nephropathy (13), focal segmental glomerulosclerosis (15), diabetic nephropathy (14), and six patients with nonglomerular diseases. Therefore, we suggest that the LG3 fragment of endorepellin could be associated with IgAN severity and might be related to pathogenesis of IgAN.

Improving the selectivity of the phosphoric acid ß-elimination on a biotinylated phosphopeptide.

This study aims at improving the MALDI-TOF detection of a phosphorylated peptide containing a cysteine residue by ß-elimination of H(3)PO(4) hardly enriched by classical methods. The experimental conditions were optimized on this phosphopeptide (biot-pAdd) and its nonphosphorylated counterpart (biot-Add). The major side-reactions were H(2)S elimination on the cysteine residues and H(2)O elimination on the non phosphorylated serine residue of biot-Add. The former dilutes the MALDI-TOF signal for the desired species. The latter gives a product similar to what is obtained by H(3)PO(4) elimination and should prompt to caution when working with a mixture between phosphorylated and non phosphorylated peptides. Modifications on the solvent, the reaction temperature and time, the nature, and concentration of the base were made. Major improvement of the selectivity of the reaction was observed in 30 % ACN, at room temperature for 4 h. However, these optimizations are specific to these sequences and should be performed anew for different peptides. The selectivity of the reaction towards H(3)PO(4) elimination is improved, but the persistence of side-reactions renders a previous sample fractionation necessary. In these optimized conditions, the ionization enhancement is 3-fold and the detection limits for biot-pAdd are similar to biot-Add (100 fmol).

Calpains contribute to vascular repair in rapidly progressive form of glomerulonephritis: potential role of their externalization.

OBJECTIVE: Calpains, calcium-activated proteases, mediate the angiogenic signals of vascular endothelial growth factor. However, their involvement in vascular repair has not been investigated and the underlying mechanisms remain to be fully elucidated. METHODS AND RESULTS: A rapidly progressive form of glomerulonephritis in wild type and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor, was studied. Calpastatin transgene expression prevented the repair of peritubular capillaries and the recovery of renal function, limiting mouse survival. In vitro analysis detected a significant reduction of both intracellular and extracellular calpain activities in transgene expressing cells, whereas Western blotting revealed that proangiogenic factors vascular endothelial growth factor and norepinephrine increased calpain exteriorization. In vitro, extracellular calpains increased endothelial cell proliferation, migration and capillary tube formation. In vivo, delivery of nonpermeable extracellular calpastatin was sufficient to blunt angiogenesis and vascular repair. Endothelial cell response to extracellular calpains was associated with fibronectin cleavage, generating fibronectin fragments with proangiogenic capacity. In vivo, fibronectin cleavage was limited in the kidney of calpastatin transgenic mice with nephritis. CONCLUSIONS: This study demonstrates that externalized calpains participate in angiogenesis and vascular repair, partly by promoting fibronectin cleavage and thereby amplifying vascular endothelial growth factor efficiency. Thus, manipulation of calpain externalization may have therapeutic implications to control angiogenesis.