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In the search for alternative inert surfaces replacing silicon chips in Desorption/Ionization On porous Silicon (DIOS)-like mass spectrometry analyses, nanostructured silicon-based NALDI™ chips were evaluated in Laser Desorption/Ionization (LDI) of peptides. Comparisons were made using commercially available DIOS chips (MassPREP™-DIOS-target™), amorphous carbon powder from lead pencil and porous silica gel used for chromatographic purposes as reference supports. A set of synthetic model peptides presenting variable amino acid sequences of various lengths was analyzed under all conditions. The LDI responses of the four ‘matrix-free’ techniques were compared, especially in terms of peptide detection sensitivity and overall experiment robustness. Copyright © 2009 John Wiley & Sons, Ltd.

Resol type resins were prepared in alkaline conditions (potassium hydroxide or potassium carbonate) using furfural obtained by acid hydrolysis of abundant renewable resources from agricultural and forestry waste residues. The structures of the resins were fully determined by 1H, 13C, and 2D NMR spectrometries with the help of four models compounds synthesized specially for this study. MALDI-Tof mass spectrometry experiments indicated that a majority of linear oligomers and a minority of cyclic ones constituted them. Composites were prepared with furfural–phenol resins and sisal fibers. These fibers were chosen mainly because they came from natural lignocellulosic material and they presented excellent mechanical properties. Thermal analyses (dTG and DSC) and electron microscopy images indicated that the composites displayed excellent adhesion between resin and fibers. Impact strength measurement showed that mild conditions were more suitable to prepare thermosets. Nevertheless, mild conditions induced a high-diffusion coefficient for water absorption by composites. Composites with good properties could be prepared using high proportion of materials obtained from biomass without formaldehyde. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Macromolecules of tunable solubility, used to mimic inert insoluble materials while maintaining solution conditions, allowed the performance of efficient supported organic chemistry and facilitated in situ reaction monitoring. To satisfy the high throughput requirements of automated synthetic processes, organic syntheses carried out on bifunctional polyethylene glycol polymers (PEG(3400)-OH) were monitored step-by-step by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A protocol was designed to control the ionization mechanism of such polymers exhibiting high affinity for alkali metal cations. Automated, rapid, and reliable data interpretation was performed by an in-house developed visual basic application relying on the sodiated ion accurate monoisotopic mass measurement. The methodology was illustrated through the monitoring of a six-step synthetic scheme.

The metabolic pathways contributing to phosphatidylcholine biosyntheses in Plasmodium falciparum, the malaria-causing parasite, was explored by electrospray ionization mass spectrometry. Phosphatidylcholine produced by the CDP-choline pathway and by the methylation of phosphatidylethanolamine was identified and quantified through isotopic labeling experiments. A straightforward method based on cone voltage directed in-source fragmentations and relative abundance measurement of endogenous versus deuterated specific fragment ions was developed for simple and rapid automated data acquisition. Such high-throughput analytical protocol allowed us to measure the relative contribution of two different metabolic pathways leading to phosphatidylcholine without performing technically more demanding and time-consuming MS/MS or LC/MS experiments.

The synthesis of a modified pentapeptide involving the palmitoylation of the hydroxyl group of a serine residue present at the N-terminal position is presented. An O–N-acyl shift was observed by LC/MS/MS, the two isobaric molecules exhibiting upon collisional activation dissociation (CAD) different fragmentation behaviours. The synthetic pathway was thereafter modified to control the palmitoylation site (O or N). The method was validated with another serine acylation (octanoylation). The evidenced mass spectrometric criteria could serve to decipher peptide post-translational modifications in proteomics.

As part of a project that aims to discover novel inhibitors of the enzymes iron- superoxide dismutases (FeSODs), a library of 4096 N t e r -acetylated C t e r -amidated tripeptides was designed. To quickly and efficiently synthesize this library without any automation or expensive techniques, an original approach combining "sort and combine" and mixture syntheses was set up. For this purpose, rink amide Synphase Lanterns were chosen as pellicular solid support equipped with colour tagging systems. 512 discrete mixtures of eight tripeptides were prepared with only 64 Synphase D-Lanterns as starting materials. All the 512 mixtures were analyzed by ESI mass spectrometry Q-Tof in the positive mode showing that all the 4096 expected members of the library were present in comparable abundances with no trace of side-products. This paper demonstrates the usefulness of tagged and divisible Synphase lanterns allowing rapid and cheap generation of large organized discrete mixture libraries with high reliability.

In the case of a soluble polymer supported synthesis, the tuning of the support solubility depending on the solvent offers new opportunities for combinatorial chemistry for two reasons. First, organic syntheses in solution are generally easily translated on a soluble polymer supported substrate. Second, complete automation of the three successive steps of combinatorial chemistry (synthesis, analytical control, biological test) is approached without releasing the synthesized compound from the support. We report in this paper the preliminary results toward automation of both soluble polymer supported syntheses and their subsequent monitoring by ESI mass spectrometry.

Heterocyclic compounds of general formula C++2Br− were studied by tandem mass spectrometry. Fragmentations taking place from the [CH]+ and [CBr]+ ions allowed identification of their structures. The two fundamental data of mass spectrometry, mass and charge, have been employed for characterization purposes.

International audience; We report applications of new hybrid organic-inorganic silica based materials as laser desorption/ionization (LDI)-promoting surfaces for high-throughput identification of peptides. The driving force of our work was to design a new material composed of a conventional MALDI matrix covalently attached to silica with a high organic/inorganic ratio in order to improve the UV absorption by such LDI hybrid matrices. Amorphous CHCA-functionalized silica presenting an organic content up to 1.3 mmol g 1 (around 40% in weight from TGA and elementary analysis measurements) gave very interesting LDI performances in terms of detection sensitivity as well as relative ionization discrepancy (spectral suppression) through the analyses of small synthetic peptide mixtures (550-1300 Da) taking CHCA and amorphous silica as model matrices for control experiments.

Insights into the direct monitoring of supported peptide synthesis were realized through the design of time of flight static secondary ion mass spectrometry (TOF-S-SIMS) experiments. The mass spectrometric method was carried out at the resin bead level and was found reproducible (intra- and inter-day assays), sensitive (femtomol level) and non-destructive (only 0.01% of the peptides were destroyed by the primary ion beam bombardment). The nature of the peptide–resin linkage governed the recovery of ions characterizing the whole peptide sequence. A S-SIMS cleavable bond was thus required solely in that position to achieve the release of the growing structures from the insoluble support into the gas phase without any fragmentation. Results are presented with standard solid-phase resins allowing linkage through an amide or an ester bond. The latter was orthogonally broken upon the bombardment and thus constituted a convenient S-SIMS cleavable bond.

International audience; Mass spectrometry is a powerful analytical tool allowing rapid and sensitive structural elucidation of a wide range of molecules issued from solution-, solid- and liquid-phase syntheses. Therefore, mass spectrometry has become the most widely used tool to probe combinatorial libraries. A significant portion of the reported combinatorial data are being produced using solid phase organic synthesis. In contrast to indirect strategies where the tethered structures were released from the support into solution to undergo standard mass spectrometric analyses, static - secondary ion mass spectrometry (S-SIMS) has enabled the identification of support-bound molecules without any chemical treatment of the resin bead. Such non-destructive characterization was applied at the bead level and facilitated the step-by-step monitoring of solid-phase peptide syntheses. Side-reactions were also detected. The relevance of S-SIMS in the rehearsal phase of combinatorial chemistry is demonstrated by comparison with infrared and nuclear magnetic resonance (NMR) spectroscopies, the two other techniques investigated in that field. An alternative to solid-phase synthesis consists of assembling molecules on a soluble polymer. This methodology is termed liquid-phase synthesis. Compound characterization is facilitated since the derivatized support is soluble in spectroscopic solvents used in NMR or in electrospray ionization mass spectrometry. The advantages and drawbacks of this approach will be discussed in terms of the direct monitoring of supported reactions during chemistry optimization and rehearsal library validation.

The use of meta-nitrobenzyl alcohol (NBA) as matrix allows to obtain better spectra than those recorded with the more popular matrix glycerol (G) in the analysis of fulvene derivatives by FAB mass spectrometry. The influence of NBA on the mass spectra corresponds to its oxidative properties. Finally, the necessary structural information was easily obtained through tandem mass spectrometry experiments.

Cyclopentapeptides containing the Arg-Gly-Asp motif have been synthesised using solid-phase assembly of side-chain-protected linear precursors, followed by solution-phase cyclisation. The replacement of the Asp residue by γ-carboxyglutamic acid (Gla) is a novel feature which gives rise to an analogue which inhibits cell adhesion, yet its congeners do not show activity in binding assays with recombinant integrin receptors. NMR techniques support a β/γ-turn conformation in most of the analogues.

During the control of a multistep organic synthesis on a soluble polymer (PEG) by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, a chemical reactivity was encountered when the matrix was acidic, for the samples where the amino moiety of the anchored compounds was protected as a Schiff base. Such imine hydrolysis was proven to be solely mediated by the acidic matrix during analyses since the expected protected structures were detected when the experiments were duplicated with a non-acidic matrix. Even if MALDI mass spectrometry was found to be more convenient than electrospray ionization mass spectrometry for the monitoring of liquid phase organic syntheses, the chemical reactivity imparted by the use of a matrix must be taken into account to avoid erroneous spectra interpretations. Copyright © 1999 John Wiley & Sons, Ltd.

Collisionally activated dissociation (CAD) spectra of protonated molecules of cyclopeptides and cyclodepsipeptides obtained with two different mass spectrometry systems were compared. Fragmentations were obtained either from collisions induced in the ion source of an electrospray mass spectrometer fitted with a single quadrupole by increasing the extracting cone voltage or from collisions with an inert gas in a free-field area of a fast-atom bombardment (FAB) mass spectrometer. Similar fragmentation pathways were produced with the two configurations even though actual tandem mass spectrometry experiments with magnetic and electric sectors provided more information than cone-induced dissociations. However, only the latter mode allowed us to perform mass spectrometric analyses coupled to liquid chromatography (LC/ESI-MS) at low cost on commercially widespread instruments. Copyright © 1999 John Wiley & Sons, Ltd.

The TOF-SIMS (time-of-flight- secondary ion mass spectrometry) spectra of Fmoc-amino acids linked to solid supports through ionic interactions have been studied to examine if easy desorption of preformed ions from the surface occurred. Preliminary results indicated that the structure of the polymer affected the nature of the recovered ions. However, in the case of widely used polystyrene based supports, characteristic ions of the amino acids were clearly observed with noticeable abundances. Moreover, an analytical criterion to differentiate Fmoc-amino acids linked to various solid supports through either covalent or ionic bonds was established. © 1998 John Wiley & Sons, Ltd.

The nature of the matrix used in Fast Atom Bombardment (FAB) mass spectrometry analyses of pyrazolo[1,2-a]pyrazoles was found to influence significantly their positive and negative ions mass spectra. Indeed the use of glycerol provided an abundant ion corresponding to the protonated molecule (M+H)+ whereas the meta-nitrobenzyl alcohol favored the formation of the radical ion M+°. Such results which are in accordance with the oxidoreduction properties of the matrices studied were also established in Frit-FAB mass spectrometry analyses of pyrazolo[1,2-a]pyrazoles.

In the rapidly growing field of combinatorial chemistry, the Multipin approach has been used for rapid and efficient multiple parallel syntheses of organic compounds. This strategy is particularly well adapted for the optimization of reaction conditions prior to chemical library syntheses, for the preparation of a wide range of compounds in relation to a lead structure or for the generation of parallel libraries involving complex multistep chemistries. In all cases, direct in situ monitoring of support-bound products would be highly valuable. In this work, time-of-flight secondary ion mass spectrometry was applied to the analysis of support-bound intermediates. The analytical method was applied to a range of crowns loaded with various dipeptides. The solid support was subjected to primary ion bombardment and characteristic ions indicative of both the peptide chain and the polymeric support were unambiguously identified. Analysis could be performed at any stage of the synthesis, and as the method is effectively non-destructive, the analyzed crowns could be further used to prepare target compounds. © 1998 John Wiley & Sons, Ltd.

A series of N,N′-linked 1,2-ethanediyl-poly(benzimidazolin-2-ones) that can be classified as podands, are described. Seven dimers (21–27), three trimers (28–30), a cyclic tetramer (31) and an open-chain benzimidazolin-2-one heptamer (32) have been prepared and characterized by 1H, 13C NMR and by MS. The crystal and molecular structure of the trimer, 1,3-bis-2-(2-oxobenzimidazol-1-yl)ethyl-2-oxobenzimidazole monohydrate (30) has been solved by X-Ray analysis. The benzi-midazolin-2-one fragments display a pattern of bond distances and angles similar to that of analogue derivatives retrieved form the CSD. The molecules are hydrogen bonded to four others in a continuous two-dimensional network by NH…OC and CH…OC interactions in which all NH donors and OC acceptors are involved.

We report the results obtained in the step by step control of a solid phase peptide synthesis carried out on Sheppard polyamide resin by Time of flight - Secondary Ion Mass Spectrometry. © 1997 Published by Elsevier Science Ltd.

Significant information on the structure of various bisazolium salts of the type CA2 (C++ 2A−) were revealed by the study of the FAB spectra of their positive and negative ions. Thus only one spectrum, whatever it consists of the positive or of the negative ions spectrum, enables the identification of the cation C++ and of the anion A−. Moreover, the same information are collected from the CAD spectrum of the adduct ion CA+ allowing the identification of such a compound in a mixture.

A straightforward method for the preparation of hybrid bioorganic–inorganic materials is reported. Common strategies to synthesize such promising materials require special surface modifications of silica followed by grafting of the organic moiety via chemoselective ligation. In this context, we set up a general and bottom-up strategy relying on modified peptides functionalized with a trialkoxysilane group. Used in mixtures with TEOS and a surfactant as the structure directing agent, these hybrid building blocks allow one step direct synthesis of bioorganic–inorganic hybrid materials. Two examples were chosen to demonstrate our general approach. (1) An antifouling surface was prepared by dip coating of a sol containing an antibacterial silylated peptide. (2) Organized mesoporous silica displaying a peptide catalyst in the pores was prepared in one step and tested.

EAPB0503 , belonging to the imidazo[1,2- a ]quinoxaline series, is an anticancer drug with antitumoral activity against a variety of tumors. Previous studies have shown that this drug undergoes demethylation and oxygenation reactions. In this paper, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was employed to assess the structures of unknown oxygenated metabolites of EAPB0503 . EAPB0503 and its identified demethylated metabolites, EAPB0502 and EAPB0603 , were incubated with human, rat, dog and mouse liver microsomes, as well as human, rat and dog hepatocytes. After separation on a C8 analytical column with a gradient elution of acetonitrile-formate buffer, positive ESI-MS/MS experiments were performed. To facilitate metabolite identification, the detailed fragmentation pathways of the parent compounds were first studied using high-resolution MS/MS. Additional hydrogen/deuterium exchange LC–MS/MS experiments were used to support the identification and structural characterization of metabolites. Four hydroxylated metabolites were identified: M′4 and its demethylated derivative M′1 (OH in ortho position on the phenyl substituent in position 1), and M′6 and its demethylated derivative M′3 (OH on the imidazole ring at the C2 position). Three phase II metabolites (Met A, EAPB0602 glucuronide; Met B, M′4 glucuronide; Met C, EAPB0603 glucuronide) were also evidenced. Elucidation of the metabolite structures was performed by comparing the chromatographic behaviors (changes in retention times), by measuring the molecular masses (mass increment), by studying the MS 2 spectral patterns of metabolites with those of parent drugs and for M′1 and M′4 by co-analysis with synthetic standards. The results of the present study provided important structural information relating to the metabolism of EAPB0503 .

International audience; A straightforward synthesis of comb-polypeptides of repeated peptide sequences was developed. These polypeptides were obtained by ROP of defined NCA without any post-polymerization grafting. The key to this strategy relies on the preparation of pure NCA bearing a peptide sequence on its side chain, by an original solid supported methodology.

cited By 8; International audience; Despite correct purity of crude peptides prepared on trityl resin by Fmoc/tBu microwave assisted solid phase peptide synthesis, surprisingly, lower yields than those expected were obtained while preparing C-terminal acid peptides. This could be explained by cyclization/cleavage through diketopiperazine formation during the second amino acid deprotection and third amino acid coupling. However, we provide here evidence that this is not the case and that this yield loss was due to high temperature promoted hydrolysis of the 2-chlorotrityl ester, yielding premature cleavage of the C-terminal acid peptides. © 2013 Springer-Verlag Wien.

International audience; The straightforward synthesis of a double-lasso macrocycle from a nonsymmetrical [c2]daisy chain, using the copper(I)-catalyzed Huisgen alkyne azide 1,3-dipolar cycloaddition, is described. The preparation of the nonsymmetrical alkyne azide [c2]daisy chain precursor was realized in situ via the exchange of the monomers contained in both symmetrical alkyne and azide [c2]daisy chains and was followed by mass spectrometry.

International audience; In the search of new robust and environmental-friendly analytical methods able to answer quantitative issues in pharmacology, we explore liquid chromatography (LC) associated with elemental mass spectrometry (ICP-MS) to monitor peptides in such complex biological matrices. The novelty is to use mass spectrometry to replace radiolabelling and radioactivity measurements, which represent up-to now the gold standard to measure organic compound concentrations in life science. As a proof of concept, we choose the vasopressin (AVP)/V1A receptor system for model pharmacological assays. The capacity of ICP-MS to provide highly sensitive quantitation of metallic and hetero elements, whatever the sample medium, prompted us to investigate this technique in combination with appropriate labelling of the peptide of interest. Selenium, that is scarcely present in biological media, was selected as a good compromise between ICP-MS response, covalent tagging ability using conventional sulfur chemistry and peptide detection specificity. Applying selenium monitoring by elemental mass spectrometry in pharmacology is challenging due to the very high salt content and organic material complexity of the samples that produces polyatomic aggregates and thus potentially mass interferences with selenium detection. Hyphenation with a chromatographic separation was found compulsory. Noteworthy, we aimed to develop a straightforward quantitative protocol that can be performed in any laboratory equipped with a standard macrobore LC-ICP-MS system, in order to avoid time-consuming sample treatment or special implementation of instrumental set-up, while allowing efficient suppression of all mass interferences to reach the targeted sensitivity. Significantly, a quantification limit of 57 ng Se L-1 (72 femtomoles of injected Se) was achieved, the samples issued from the pharmacological assays being directly introduced into the LC-ICP-MS system. The established method was successfully validated and applied to the measurement of the vasopressin ligand affinity for its V1A receptor through the determination of the dissociation constant (Kd) which was compared to the one recorded with conventional radioactivity assays.

International audience; By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (bn-1+H2O) ion, corresponding to a shortened nonscrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H2OandNH3) and multiple backbone amide ruptures (b-type internal ions), the described Cterminal residue exclusion is highly identifiable giving raise to a single fragment ion in the high mass range of the MS/MS spectra. The mass difference between this signal and the protonated molecular ion corresponds to the mass of the C-terminal residue. It allowed a straightforward identification of the amino acid positioned at this extremity. It must be emphasized that a neutral residue loss can be misattributed to the formation of a ym-1 ion, i.e., to the loss of the N-terminal residue following the a1ym-1 fragmentation channel. Extreme caution must be adopted when reading the direct sequence ion on the positive ion MS/MS spectra of singly charged peptides not to mix up the attribution of the N- and C-terminal amino acids. Although such peculiar fragmentation behavior is of obvious interest for de novo peptide sequencing, it can also be exploited in proteomics, especially for studies involving digestion protocols carried out with proteolytic enzymes other than trypsin (Lys-N, Glu-C, and Asp-N) that produce arginine-containing peptides.

International audience; The present study was conducted to assess the structures of the main unknown oxygenated metabolites of EAPB0203. The first step was to assign all the 1H and 13C NMR of both EAPB0203 and its demethylated metabolite (EAPB0202) to the corresponding atoms in their molecular structures and to elucidate the fragmentation pathways for the [M + H] + ions of these compounds using high-resolution mass spectrometry (MS). MS/MS spectra showed that both protonated molecules possessing an even number of electrons were unexpectedly losing radicals such as H*,CH3*, or even C7H7* giving stable radical cations. In vitro metabolism studies were investigated in rat and dog liver microsomes and in the filamentous fungus Cunninghamella elegans. Structural elucidation of six oxygenated metabolites was performed based on the following: (i) their fragmentation pathways in liquid chromatography−MS/MS (LC-MS/MS) analyses; (ii) comparison of their changes in their molecular masses and fragment ions with those of the parent drugs; and (iii) the results of online H/D exchange experiments that provided additional evidence in differentiating hydoxylated metabolites from N-oxides. Structures of the metabolites were elucidated by LC-MS/MS and comparison with synthetic standards; structures of these standards were confirmed using one- and two-dimensional 1H NMR spectroscopies.

International audience; Sulfation of tyrosine residues is a key posttranslationalmodification in the regulation of various cellular processes. As such, the detection and localization of tyrosine sulfation is an essential step toward the elucidation of the physiological and pathological roles of this process. Despite substantial advances, intact sulfated peptides are still difficult to detect by MALDI-MS due to the extreme lability of the sulfo-moiety. The present report demonstrates for the first time how intact sulfated peptides can be directly and specifically detected by MALDI-MS in positive reflectronmode by using pyrenemethylguanidine (pmg) as a noncovalent derivatizing agent and an ionization enhancer. This new method allows the determination of the degree of sulfation of sulfopeptides pure or in mixtures. Moreover, the observation of specific peaks in the mass spectra enables a rapid and unambiguous discrimination between phospho- and sulfopeptides.

We report on the simple deposition of Stober silica nanoparticles (SiO 2 NPs) on conventional MALDI target plate for high throughput laser desorption/ionization mass spectrometry (LDI-MS) analyses of peptide mixtures with sensitivity in the femtomolar range. This low-cost easily prepared material allowed straightforward LDI experiments by deposition of the studied samples directly onto a pre-spotted MALDI plate. This analytical strategy can be performed in any laboratory equipped with a MALDI-TOF instrument. All key benefits of organic matrix-free technologies were satisfied while maintaining a high level of detection performances (sensitivity and reproducibility/repeatability). In particular, sample preparation was simple and detection in the low mass range was not hampered by matrix ions. Imaging studies were undertaken to query sample dispersion into the inert SiO 2 NPs and to help into the search of the best experimental conditions producing homogeneous analyte distribution within the deposit. In contrast to commercial disposable LDI targets designed for single use and requiring an adaptor such as NALDI™, the proposed SiO 2 NPs pre-spotting on a MALDI target plate allowed very easily switching between MALDI and LDI experiments. They can be conducted either simultaneously (positions with an organic matrix or SiO 2 NPs) or in the row (support prepared in advance, stored and washed after use). The overall cost and versatility of the methodology made it very attractive to MALDI users in many domains (peptidomics, proteomics, metabolomics).

cited By 3; International audience; The synthesis and use of novel polymer-supported reagents for disulfide bond formation is described. This family of supported reagents consists of a series of oxidized methionines grafted onto a solid support. Their cost and the simplicity of their preparation through N-carboxyanhydride polymerization on beads make them reactants of choice for the formation of disulfide bridges in peptides. Copyright © 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

International audience; In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola Frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion since a specific cleavage at the amide X-Lys chain is obtained providing N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a In lysine or arginine residues solely at the C-terminal position, and thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with ESI-QqTof and MALDI-Tof/Tof mass spectrometers commonly used in proteomic bottom-up experiments were investigated.

International audience; In this paper, mesoporous organosilicas functionalised with aminopropyl groups have been successfully used for peptide oligomerisation. For this purpose, three mesoporous silica SBA-15 type containing different amounts of aminopropyl groups were prepared by direct synthesis and using 3- tert-butyloxycarbonylamino propyltriethoxysilane. Thanks to amino groups and under well selected experimental conditions, amino acids N-carboxyanhydride-polymerisation has been achieved within the pores with control of the physical properties of peptide functionalised hybrid materials. NCA of alanine, side chain protected glutamic acid and methionine were used for this study. For the first time, direct LDI-MS analysis was successfully performed on the resulting covalently bound supported oligomers. To demonstrate the potential application of this class of hybrid bio-organic-inorganic material as supported catalysts, one of the methionine-functionalized OMSs was used to promote disulfide bond formation in a model peptide.

International audience; A wide range of chemical reagents are available to study the protein-protein interactions or protein structures. After reaction with such chemicals, covalently modified proteins are digested, resulting in shorter peptides that are analyzed by mass spectrometry (MS). Used especially when NMR of X-ray data are lacking, this methodology requires the identification of modified species carrying relevant information, among the unmodified peptides. To overcome the drawbacks of existing methods, we propose a more direct strategy relying on the synthesis of solid-supported cleavable monofunctional reagents and cross-linkers that react with proteins and that selectively release, after protein digestion and washings, the modified peptide fragments ready for MS analysis. Using this Solid-Phase Cross-Linking (SPCL) strategy, only modified sequences are analyzed and consistent data can be easily obtained since the signals of interest are not masked or suppressed by over-represented unmodified materials.

International audience; The Pipecolic linker is a new highly versatile handle which immobilizes on solid support through a carboxylic acid function a wide range of amines, alcohols, and hydrazines. The anchoring step on pipecolic resin is very easy and efficient, and compounds are released with high purities upon acidic treatment. During this treatment, an oxazolonium intermediate is hydrolyzed, yielding the cleavage of ester or amide bond and the release of free carboxylic acid of the starting linker. In this study, we report the possibility of recycling the pipecolic resin after the use of several trifluoroacetic acid (TFA) cleavage cocktails. We demonstrate that it can be reused up to five times without significant loading decrease.

The selective removal of N-Boc protection can be obtained in the presence of either TBDMS or TBDPS ethers. On the basis of promising results from the literature, we first tried sonication, that failed, whereas the exclusive cleavage of the Boc group was successfully achieved by a saturated solution of HCl in ethyl acetate.

The nature of the matrix used in Fast Atom Bombardment (FAB) mass spectrometry analyses of pyrazolo[1,2-a]pyrazoles was found to influence significantly their positive and negative ions mass spectra. Indeed the use of glycerol provided an abundant ion corresponding to the protonated molecule (M+H)+ whereas the meta-nitrobenzyl alcohol favored the formation of the radical ion M+°. Such results which are in accordance with the oxidoreduction properties of the matrices studied were also established in Frit-FAB mass spectrometry analyses of pyrazolo[1,2-a]pyrazoles.

Mass spectrometry is a powerful analytical tool allowing rapid and sensitive structural elucidation of a wide range of molecules issued from solution-, solid- and liquid-phase syntheses. Therefore, mass spectrometry has become the most widely used tool to probe combinatorial libraries. A significant portion of the reported combinatorial data are being produced using solid phase organic synthesis. In contrast to indirect strategies where the tethered structures were released from the support into solution to undergo standard mass spectrometric analyses, static - secondary ion mass spectrometry (S-SIMS) has enabled the identification of support-bound molecules without any chemical treatment of the resin bead. Such non-destructive characterization was applied at the bead level and facilitated the step-by- step monitoring of solid-phase peptide syntheses. Side-reactions were also detected. The relevance of S-SIMS in the rehearsal phase of combinatorial chemistry is demonstrated by comparison with infrared and nuclear magnetic resonance (NMR) spectroscopies, the two other techniques investigated in that field. An alternative to solid-phase synthesis consists of assembling molecules on a soluble polymer. This methodology is termed liquid-phase synthesis. Compound characterization is facilitated since the derivatized support is soluble in spectroscopic solvents used in NMR or in electrospray ionization mass spectrometry. The advantages and drawbacks of this approach will be discussed in terms of the direct monitoring of supported reactions during chemistry optimization and rehearsal library validation.

International audience; Deinococcaceae are a family of extremely radiation tolerant bacteria that is currently subjected to numerous studies aimed at understanding the molecular mechanisms for such radiotolerance. To achieve a comprehensive and accurate annotation of Deinococcus deserti genome, we performed an N-terminal-oriented characterization of its proteome. For this, we used a labeling reagent, N-Tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP), to selectively derivatize protein N-termini. The large scale identification of TMPP-modified Nterminal- most peptides by a shotgun liquid chromatography-tandem mass spectrometry analysis led to the validation of 278 and correction of 73 translation initiation codons in D. deserti genome. In addition, four new genes were detected, three located on the main chromosome and one on plasmid P3. Surprisingly, experimental evidences were obtained indicating that DnaA (the protein involved in the DNA replication initiation process) and RpsL (the S12 ribosomal conserved protein) translation is initiated in Deinococcaceae from non-canonical codons (ATC and CTG, respectively). Such use may be the basis of specific regulation mechanisms affecting replication and translation. We report the use of nonconventional translation initiation codons for two other genes: Deide_03051 and infC. We also analyzed signal peptide cleavages at a genome-wide scale. Based on comparative proteogenomic analysis, we propose a set of 137 corrections to improve Deinococcus radiodurans and Deinococcus geothermalis gene annotations. Some of these corrections affect important genes involved in DNA repair mechanisms such as polA, ligA, and ddrB. Our results demonstrate that predicting translation initiation codons is still difficult and that proteomic-based refinement of genome annotations is necessary. Whether such use of noncanonical translation initiation codons is much more frequent for other bacterial phyla than previously reported or restricted to Deinococcaceae remains to be investigated. Our results demonstrate that predicting translation initiation codons is still difficult for some bacteria and that proteomics-based refinement of genome annotations may be helpful in such cases.

International audience

International audience; We report our contribution to the systematic investigation of peptide fragmentations performed on high-performance Tof equipement operating in MS and MS/MS modes, such as ESI-QqTof and MALDI-Tof/Tof intruments that are commonly available today in proteomic laboratories. Whereas the former analyzer's configuration provides low-energy collision-induced dissociations (CID), the latter allows tunable activation methods of the selected parent ion to induce either metastable laser-induced dissociations (LID) or high-energy CID ('gas on spectra LID'). Fragmentation of the monoprotonated ion of 53 peptides (FW 807-2853 g/mol) was undertaken upon low-energy CID on an ESI-QT of mass spectrometer (Waters) as well as high-energy CID and LID conditions on a MALDI Ultraflex mass spectrometer (Bruker). Systematic comparison of MS/MS spectra provided useful information on the performance of each piece of equipment for efficient peptide sequencing and also insights into the observed fragmentation behaviors.

154MF Times Cited:4 Cited References Count:51; Efficient peptide sequencing relies on both high quality MS/MS data acquisition and exhaustive knowledge of gas-phase dissociation mechanisms. We report our contribution to the elaboration of more comprehensive fragmentation models required for efficient automated MS/MS spectra interpretation. Following a statistical approach, various peptides (296 sequences of variable compositions and lengths) were prepared and subjected to low-energy collision-induced dissociations (CID) in an electrospray hybrid instrument (ESI-Q-q-Tof type mass spectrometer) that has retained relatively limited attention so far. Besides, our studies were focused on low molecular weight singly charged peptides that often failed to be identified by sequencing algorithms. Only half of the studied compounds showed charge directed dissociations in accordance with the mobile proton model producing fragment ions directly related to the primary sequence. For the peptides that did not exhibit the expected fragment ion series, alternative dissociation behaviors issued from complex rearrangements were evidenced.

The behavior of C-terminal amidated and carboxylated peptides upon low-energy collision-induced dissociation (CID) was investigated. Two sets of 76 sequences of variable amino acid compositions and lengths were synthesized as model compounds. In most cases, C-terminal amidated peptides were found to produce, upon CID, an abundant loss of ammonia from the protonated molecules. To validate such MS/MS signatures, the studied peptides contained amino acids that can potentially release ammonia from their side chains, such as asparagine, glutamine, tryptophan, lysine and arginine. Arginine, and to a lesser extent lysine, was shown to induce a competitive fragmentation leading to the loss of ammonia from their side chains, thus interfering with the targeted backbone neutral release. However, when arginine or lysine was located at the C-terminal position mimicking a tryptic digest, losses of ammonia from the arginine side chain and from the peptide backbone were completely suppressed. Such results were discussed in the frame of peptidomic or proteomic studies in an attempt to reveal the presence of C-terminal amidated peptides or proteins.