Light-Cleavable Auxiliary for Diselenide-Selenoester Ligations of Peptides and Proteins.

Diselenide-selenoester ligations are increasingly used for the synthesis of proteins. Excellent ligation rates, even at low concentrations, in combination with mild and selective deselenization conditions can overcome some of the most severe challenges in chemical protein synthesis. Herein, the versatile multicomponent synthesis and application of a new ligation auxiliary that combines a photocleavable scaffold with the advantages of selenium-based ligation strategies are presented. Its use was investigated with respect to different ligation junctions and describe a novel para-methoxybenzyl deprotection reaction for the selenol moiety. The glycine-based auxiliary enabled successful synthesis of the challenging target protein G-CSF.

Labeling and Natural Post-Translational Modification of Peptides and Proteins via Chemoselective Pd-Catalyzed Prenylation of Cysteine.

The prenylation of peptides and proteins is an important post-translational modification observed in vivo. We report that the Pd-catalyzed Tsuji-Trost allylation with a Pd/BIPHEPHOS catalyst system allows the allylation of Cys-containing peptides and proteins with complete chemoselectivity and high n/i regioselectivity. In contrast to recently established methods, which use non-native connections, the Pd-catalyzed prenylation produces the natural n-prenylthioether bond. In addition, a variety of biophysical probes such as affinity handles and fluorescent tags can be introduced into Cys-containing peptides and proteins. Furthermore, peptides containing two cysteine residues can be stapled or cyclized using homobifunctional allylic carbonate reagents.

Mehercules, adhuc Bacchus! The debate on wine proteomics continues.

The proteome of untreated white wines (a Recioto made with Garganega grapes from the Veneto region) was explored in depth via capture with combinatorial peptide ligand libraries (CPLL) at four different pH values: pH 2.2, 3.8, 7.2, and 9.3. The combined data on the discoveries in the four CPLL eluates, as well as in the collected bottle sediment, allowed the identification of 106 unique gene products belonging to Vitis vinifera, as well as of an additional 11 proteins released by the S. cerevisiae used in the fermentation process. Among the residual grape proteins detected in the Recioto wine, ca. 30% were categorized as medium to high-abundance species, vs 70% low-abundance ones. The detection of so many low-abundance species suggests that proteomic (coupled to peptidomic) data might be used for typing high-quality products (grand crus) to assess their genuineness and protect them from fraudulent imitations.

Going nuts for nuts? The trace proteome of a Cola drink, as detected via combinatorial peptide ligand libraries.

The "invisible" proteome of a Cola drink, stated to be produced with a kola nut extract, has been investigated via capture with combinatorial peptide ligand libraries (CPLL). Indeed, a few proteins in the M(r) 15-20 kDa range could be identified by treating large beverage volumes (1 L) and performing the capture with CPLLs at very acidic pH values (pH 2.2) under conditions mimicking reverse-phase adsorption. Ascertaining the presence of proteins deriving from plant extracts has confirmed the genuineness of such beverage and suggests the possibility of certifying whether soft drinks present on the market are indeed made with vegetable extracts or only with artificial chemical flavoring.

In-depth proteomic analysis of non-alcoholic beverages with peptide ligand libraries. I: Almond milk and orgeat syrup.

Combinatorial peptide ligand libraries, both commercial and home-made, have been adopted to investigate the proteome of non-alcoholic beverages, in order to assess their genuineness and detect also trace proteins, in search of potential allergens. Two such beverages have been studied: almond milk and orgeat syrup. In the first product we have been able to identify 132 unique protein species, the deepest investigation so far of the almond proteome. In the second beverage, a handful of proteins (just 14) have been detected, belonging to a bitter almond extract. In both cases, the genuineness of such products has been verified, as well as the fact that almond milk, judging on the total protein and fat content, must have been produced with 100g ground almonds per litre of beverage, as required by authorities. On the contrary, cheap orgeat syrups produced by local supermarkets and sold as their own brands, where found not to contain any residual proteins, suggesting that they contained only synthetic aromas and no natural plant extracts. This could be the starting point for investigating the myriad of beverages that in the last decades have invaded the shelves of supermarkets the world over, whose genuineness and natural origin have never been properly assessed.

Popeye strikes again: The deep proteome of spinach leaves.

The cytoplasmic proteome of spinach leaves (Spinacia oleracea L) has been investigated with the help of commercially available (ProteoMiner) combinatorial peptide ligand libraries and with home-made ligand beads as prepared in our laboratory. The protein capture had been performed at three pH values (4.0, 7.0 and 9.3) and elution performed in 4% boiling SDS, 20mM DTT. The total number of unique gene products identified amounts to 322 proteins, of which 114 are in common with the control, untreated sample, 18 are present only in the control and 190 represent the new species detected with the help of all combined eluates and likely represent low-abundance species. This is the first in depth exploration of the spinach cytoplasmic proteome and might enable further studies on interaction, regulation and expression of proteins biological processes in combination or not with transcriptomics data.

Noah's nectar: the proteome content of a glass of red wine.

Combinatorial peptide ligand libraries (CPLLs) have been adopted for harvesting and identifying traces of proteins present in red wines. Surprisingly, although it is stated that red wines are in general fined with egg albumin, for all Italian wines investigated (in the areas around Chiari and Verona as well as in the Chianti area) we find that the only fining agent used is bovine casein, just like in white wines. Although the typical levels of casein found range between 45 to 85µg/L, in one case as little as 3.8µg/L of casein could be detected, an extremely high level of sensitivity, close to our lower detection limit of 1µg/L reported for white wines. As a result of such treatments, very small amounts of residual proteins in red wines could be identified: essentially no residual grape proteins (except for thaumatin), but only traces of proteins from Saccharomyces cerevisiae and a few proteins from plant pathogens and fungi (e.g., Botryotinia fuckeliana, Sclerotinia sclerotiorum, Aspergillus aculeatus). Contrary to what has been found in white wines, the best capture efficiency with CPLLs has occurred at pH 7.2 and pH 9.3, with minimal capture at pH 3.3. The fact that such very low levels of fining agents can still be detected in treated red wines should be taken into consideration by winemakers in labelling their products and by EC rulers in issuing proper regulations.

Les Maîtres de l'Orge: the proteome content of your beer mug.

The beer proteome has been evaluated via prior capture with combinatorial peptide ligand libraries (ProteoMiner as well as a homemade library of reduced polydispersity) at three different pH (4.0, 7.0, and 9.3) values. Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could categorize such species in 20 different barley protein families and 2 maize proteins, the only ones that had survived the brewing process (the most abundant ones being Z-serpins and lipid transfer proteins). In addition to those, we could identify 40 unique gene products from Saccharomyces cerevisiae, one from S. bayanus and one from S. pastorianus as routinely used in the malting process for lager beer. These latter species must represent trace components, as in previous proteome investigations barely two such yeast proteins could be detected. Our protocol permits handling of very large beer volumes (liters, if needed) in a very simple and user-friendly manner and in a much reduced sample handling time. The knowledge of the residual proteome in beers might help brewers in selecting proper proteinaceous components that might enrich beer flavor and texture.

The proteome buccaneers: how to unearth your treasure chest via combinatorial peptide ligand libraries.

The latest advances in combinatorial peptide ligand libraries, with their unique performance in discovering low-abundance species in proteomes, are reviewed here. Explanations of mechanism, potential applications, capture of proteomes at different pH values to enhance the total catch and quantitative elutions, such as boiling in the presence of 5% sodium dodecyl sulfate and 3% dithiothreitol are included. The reproducibility of protein capture among different experiments with the same batch of beads or with different batches is also reported to be very high, with coefficient of variations in the order of 10-20%. Miniaturized operations, consisting of capture with as little as 20 or even 5 microl of peptide beads are reported, thus demonstrating that the described technology could be exploited for routine biomarker discovery in a biomedical environment. Finally, it is shown that the signal of captured proteins is linear over approximately three orders of magnitude, ranging from nM to microM, thus ensuring that differential quantitative proteomics for biomarker discovery can be fully implemented, providing species do not saturate their ligands.

Proteomics of wine additives: mining for the invisible via combinatorial peptide ligand libraries.

Combinatorial peptide ligand libraries (CPLLs) have been adopted for harvesting and identifying traces of casein (used as a fining agent) present in white wines. Although minute amounts (200 microL) of CPLL beads are added to the entire content of a wine bottle (750 mL), they are able to sequester with high efficiency (up to 80%) residual traces of casein, permitting a signal "amplification" of at least 5000-fold. It is here demonstrated that as little as 1 microg/L of casein can be efficiently detected in white wines, a major improvement over previous investigations in which the lower detection limit had been estimated at 100 microg/L. The fact that such very low levels of fining agents can still be detected in treated white wines should be taken into consideration by winemakers in labelling their products and by EC rulers in issuing proper regulations.

Interaction among proteins and peptide libraries in proteome analysis: pH involvement for a larger capture of species.

When capturing proteins via combinatorial peptide ligand libraries, a method well known for drastically reducing the concentration of high-abundance proteins and substantially magnifying the signal of low-abundance species, thus leading to the discovery of a large number of proteins previously undetected in proteomes, we had constantly noticed that there would be a loss of species initially present in the untreated sample, to the tune of 5%, up to 15% in some cases. Such losses are a nuisance and hamper to some extent the unique performance of the method. In order to verify if such losses could be reduced and also to understand some mechanisms of the capture process, we introduce here an important variant to the capture operation, up to the present carried out in physiological saline at pH 7.2. In this novel protocol, the binding step is conducted at three different pH values, namely the standard one at pH 7.2, plus two additional processes, at acidic (pH 4.0) and alkaline (pH 9.3) pH values. Indeed the capture process is more extensive, with a number of additional species captured at the two pH extremes in sera and other proteomes. Interestingly, at pH 4.0 newly detected proteins were mostly acidic, while at the alkaline pH additional protein species were more evenly distributed throughout the pI range towards the alkaline area. The role of pH in the complex mechanism of binding among the hexapeptide library and the various proteomes being analyzed is discussed and evaluated. Due to significant changes in protein patterns with pH, recommendations are thus made to increase the possibility to find additional gene products illustrated by two examples (snake venom and leaf protein extract). Keeping under control the environmental pH when facing reproducibility studies or for comparative proteomics profiling is also a general rule suggested by this study.

pI-based fractionation of serum proteomes versus anion exchange after enhancement of low-abundance proteins by means of peptide libraries.

The pre-treatment of biological extracts with the aim of detecting very low-abundance proteins generates complexity requiring a proper fractionation. Therefore the success of identifying all newly detectable species depends on the selected fractionation methods. In this context and starting from a human serum, where the dynamic concentration range was reduced by means of a preliminary treatment with a combinatorial hexapeptide ligand library, we fractionated the sample using a novel method based on the differences in isoelectric points of proteins by means of Solid-State Buffers (SSB) associated with cation exchangers. The number of fractions was limited to four and was compared to a classical anion exchange method generating the same number of fractions. What was observed is that when using SSB technology the protein redundancy between fractions was significantly reduced compared to ion exchange fractionation allowing thus a better detection of novel species. The analysis of trypsinized protein fractions by nanoLC-MS/MS confirmed that the SSB technology used is more discriminant than anion exchange chromatography fractionation. A sample fractionation by SSB after the reduction of dynamic concentration range can be accomplished without either adjustment of pH and ionic strength or protein concentration and cleanup. Both advantages over either classical chromatography or isoelectric fractionations allow approaching the discovery of markers of interest under easier conditions applicable in a variety of fields of investigation.

En bloc elution of proteomes from combinatorial peptide ligand libraries.

In contrast to the three to four sequential elution steps routinely adopted for recovering proteomes adsorbed onto combinatorial peptide ligand libraries, we report here two en bloc elution systems, which are able to achieve recoveries in the order of 95% in a single step. One consists of TUC (7 M urea, 2 M thiourea, 3% CHAPS) added with 40 mM formic acid, the other of TUC added with 25 mM cysteic acid (Cys-A). Although both systems are almost equally performing, the formic acid eluant has as a drawback, namely the potential to modify proteins by formylation of Ser and Thr residues. On the contrary, the Cys-A system is unreactive towards proteins. Additionally, Cys-A, due to its very low pI value (1.80) does not interfere with subsequent 2D map analyses since, during the first isoelectric focusing step (in general performed in immobilized pH gradients), it migrates to the anodic compartment and thus vacates the gel. Conversely, formic acid would mostly collect around pH 3 and acetic or citric acid, formerly used in the UCA (9 M urea, 50 mM citric acid) eluant, would condense around pH 4 in the focusing step, interfering thus with 2D map analyses. Elution by boiling SDS of the small amount of protein left over after three sequential elution steps in TUC and 25 mM Cys-A and analysis by nanoLC-MS/MS has demonstrated that these residual proteins are indeed a residue left over from proteins already eluted in TUC-Cys-A and not new species absent in the latter eluate.

Functional identification of catalytic metal ion binding sites within RNA.

The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-SP phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.

Application of Brønsted-type LFER in the study of the phospholipase C mechanism.

Phosphatidylinositol-specific phospholipase C cleaves the phosphodiester bond of phosphatidylinositol to form inositol 1,2-cyclic phosphate and diacylglycerol. This enzyme also accepts a variety of alkyl and aryl inositol phosphates as substrates, making it a suitable model enzyme for studying mechanism of phosphoryl transfer by probing the linear free-energy relationship (LFER). In this work, we conducted a study of Brønsted-type relationship (log k = beta(lg) pK(a) + C) to compare mechanisms of enzymatic and nonenzymatic reactions, confirm the earlier proposed mechanism, and assess further the role of hydrophobicity in the leaving group as a general acid-enabling factor. The observation of the high negative Brønsted coefficients for both nonenzymatic (beta(lg) = -0.65 to -0.73) and enzymatic cleavage of aryl and nonhydrophobic alkyl inositol phosphates (beta(lg) = -0.58) indicates that these reactions involve only weak general acid catalysis. In contrast, the enzymatic cleavage of hydrophobic alkyl inositol phosphates showed low negative Brønsted coefficient (beta(lg) = -0.12), indicating a small amount of the negative charge on the leaving group and efficient general acid catalysis. Overall, our results firmly support the previously postulated mechanism where hydrophobic interactions between the enzyme and remote parts of the leaving group induce an unprecedented negative-charge stabilization on the leaving group in the transition state.

Engineering a catalytic metal binding site into a calcium-independent phosphatidylinositol-specific phospholipase C leads to enhanced stereoselectivity.

Eukaryotic phosphatidylinositol-specific phospholipase Cs (PI-PLCs) utilize calcium as a cofactor during catalysis, whereas prokaryotic PI-PLCs use a spatially conserved guanidinium group from Arg69. In this study, we aimed to construct a metal-dependent mutant of a bacterial PI-PLC and characterize the catalytic role of the metal ion, seeking an enhanced understanding of the functional differences between these two positively charged moieties. The following results indicate that a bona fide catalytic metal binding site was created by the single arginine-to-aspartate mutation at position 69: (1) The R69D mutant was activated by Ca(2+), and the activation was specific for R69D, not for other mutants at this position. (2) Titration of R69D with Ca(2+), monitored by (15)N-(1)H HSQC (heteronuclear single quantum coherence) NMR, showed that addition of Ca(2+) to R69D restores the environment of the catalytic site analogous to that attained by the WT enzyme. (3) Upon Ca(2+) activation, the thio effect of the S(P)-isomer of the phosphorothioate analogue (k(O)/k(Sp) = 4.4 x 10(5)) approached a value similar to that of the WT enzyme, suggesting a structural and functional similarity between the two positively charged moieties (Arg69 and Asp69-Ca(2+)). The R(P)-thio effect (k(O)/k(Rp) = 9.4) is smaller than that of the WT enzyme by a factor of 5. Consequently, R69D-Ca(2+) displays higher stereoselectivity (k(Rp)/k(Sp) = 47,000) than WT (k(Rp)/k(Sp) = 7600). (4) Results from additional mutagenesis analyses suggest that the Ca(2+) binding site is comprised of side chains from Asp33, Asp67, Asp69, and Glu117. Our studies provide new insight into the mechanism of metal-dependent and metal-independent PI-PLCs.