Cysteinyl Peptides Research Articles

Differentiation of isobaric cross-linked peptides prepared via maleimide chemistry using MALDI-MS and MS/MS.

The thiosuccinimide linker is widely used in the synthesis of bioconjugates. However, it is susceptible to hydrolysis and is transformed into its hydrolyzed and/or the isobaric thiazine forms, the latter of which is a fairly common product in a conjugate that contains a cysteinyl peptide. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS) are useful for differentiating these isobaric species. Four cross-linked peptides with thiosuccinimide linkers were synthesized. Analogs with linkers that were transformed into thiazine and/or the hydrolyzed thiosuccinimide linkers were then synthesized by incubating the samples at neutral or basic pH. All the cross-linked peptides were purified using RP-HPLC (reversed-phase high-performance liquid chromatography) and differentiated using MALDI-MS, MALDI-MS/MS, and ultraviolet photodissociation. A cysteinyl peptide-containing conjugate, the thiosuccinimide form, was largely transformed into the hydrolyzed or thiazine forms after incubation at neutral or basic pH. MALDI-MS allowed the three forms to be differentiated: the thiosuccinimide and its hydrolysis product yielded two constituent peptides after reductive cleavage between the Cys and succinimide moieties; no fragment ions were produced from the thiazine form. In addition, MALDI-MS/MS of the thiosuccinimide form yielded two pairs of complementary fragment ions via 1,4-elimination: Cys-SH and maleimide, and dehydro-alanine and thiosuccinimide, which are different from those produced via reductive cleavage in MALDI-MS. The thiazine form yielded fragment ions resulting from the cleavage of the newly formed amide bond in the linker that resulted from thiazine formation. The thiosuccinimide (but not thiazine) form of the cross-linked peptide yielded individual constituent peptides using MALDI-MS and MALDI-MS/MS, showing specific 1,4-elimination for the thiosuccinimide form and cleavage at the newly formed peptide bond via transcyclization for the thiazine form.

Synthesis of S-Carbamidomethyl Cysteine and Its Use for Quantification of Cysteinyl Peptides by Targeted Proteomics.

Proteomics has played a central role in the identification of reliable disease biomarkers, which are the basis of precision medicine, a promising approach for tackling recalcitrant diseases such as cancer, that elude conventional treatments. Among proteomic methodologies, targeted proteomics employing stable isotope-labeled (SIL) internal standards is particularly suited for the clinical translation of biomarker information owing to its high throughput and accuracy in the quantitative analysis of patient-derived proteomes. Using SIL internal standards ensures the utmost level of confidence in detection and precision in targeted MS experiments. For successfully establishing assays based on targeted proteomics, it is crucial to secure broad coverage when selecting the SIL standard peptide panel. However, cysteinyl peptides have often been excluded because of cysteine's high chemical reactivity. To address this limitation, a new cysteine building block was developed by incorporating a sulfhydryl group configured with an S-carbamidomethyl group, which is commonly used in proteome sampling. This compound was found to be chemically stable and applicable to a variety of solid-phase peptide synthesis (SPPS) campaigns. Furthermore, a direct comparison of the synthesized SIL peptides and tryptic endogenous peptides demonstrated the potential utility of an SPPS flow based on the new cysteine building block for improving the success of targeted proteomic applications.

Fluorescence by self-assembly: autofluorescent peptide vesicles and fibers.

A series of oxidized cysteinyl peptides ([P-Cys-X-OMe]2; P = Boc or H; X = Trp or Glu) showed vesicular and fibrillar assemblies. The anatomy of the self-assembled vesicles from the water-soluble cystine peptide [Cys-Trp-OMe]2 (1a) has been investigated by using various fluorescent probes such as ammonium 8-anilinonaphthalene-1-sulfonate, Nile Red and pyrene. The morphological characterization was carried out by fluorescence lifetime imaging microscopy (FLIM) and super resolution-structured illumination microscopy (SR-SIM) utilizing the autofluorescence of the vesicles stemming from the self-assembly. The self-assembled structures are also observed in solution as evident from the quantitative phase images obtained using a dual-mode digital holographic microscope (DHM) system. Present investigations show that the self-assembly is enthalpy- and entropy-driven in the aqueous medium. Based on the CD spectral studies, we proposed that 1a organizes into vesicles through the sequestration of indole units. We observed that the solutions of dipeptides 1a-b exhibit autofluorescence in the blue region upon excitation at a wavelength >350 nm. Detailed spectroscopic studies on the peptides lacking tryptophan 2a-b unequivocally showed that the autofluorescence stems exclusively from peptide aggregation. Our experimental results with appropriate controls revealed that the clustering of carbonyl chromophores is central to autofluorescence. Autofluorescence was also used to probe the vesicle formation without using any external fluorescent probe. To the best of our knowledge, this is the first report on autofluorescent vesicles formed by the spontaneous association of dipeptides. We also found that the vesicles formed by 1a can act as a host for guests like C60. The biocompatibility and biodegradability of these peptides along with the autofluorescent nature and guest binding ability of peptide-based vesicles offer numerous applications in the biomedical area.

Redox-Controlled Chemical Protein Synthesis: Sundry Shades of Latency.

ConspectusThe last two decades have witnessed the rise in power of chemical protein synthesis to the point where it now constitutes an established corpus of synthetic methods efficiently complementing biological approaches. One factor explaining this spectacular evolution is the emergence of a new class of chemoselective reactions enabling the formation of native peptide bonds between two unprotected peptidic segments, also known as native ligation reactions. In recent years, their application has fueled the production of homogeneous batches of large and highly decorated protein targets with a control of their composition at the atomic level. In doing so, native ligation reactions have provided the means for successful applications in chemical biology, medicinal chemistry, materials science, and nanotechnology research.The native chemical ligation (NCL) reaction has had a major impact on the field by enabling the chemoselective formation of a native peptide bond between a C-terminal peptidyl thioester and an N-terminal cysteinyl peptide. Since its introduction in 1994, the NCL reaction has been made the object of significant improvements and its scope and limitations have been thoroughly investigated. Furthermore, the diversification of peptide segment assembly strategies has been essential to access proteins of increasing complexity and has had to overcome the challenge of controlling the reactivity of ligation partners.One hallmark of NCL is its dependency on thiol reactivity, including for its catalysis. While Nature constantly plays with the redox properties of biological thiols for the regulation of numerous biochemical pathways, such a control of reactivity is challenging to achieve in synthetic organic chemistry and, in particular, for those methods used for assembling peptide segments by chemical ligation. This Account covers the studies conducted by our group in this area. A leading theme of our research has been the conception of controllable acyl donors and cysteine surrogates that place the chemoselective formation of amide bonds by NCL-like reactions under the control of dichalcogenide-based redox systems. The dependency of the redox potential of dichalcogenide bonds on the nature of the chalcogenides involved (S, Se) has appeared as a powerful means for diversifying the systems, while allowing their sequential activation for protein synthesis. Such a control of reactivity mediated by the addition of harmless redox additives has greatly facilitated the modular and efficient preparation of multiple targets of biological relevance. Taken together, these endeavors provide a practical and robust set of methods to address synthetic challenges in chemical protein synthesis.

Superfast desulfurization for protein chemical synthesis and modification

Superfast desulfurization for protein chemical synthesis and modification

Prebiotic Environments with Mg2+ and Thiophilic Metal Ions Increase the Thermal Stability of Cysteine and Non-cysteine Peptides.

Wet-dry cycles driven by heating to high temperatures are frequently invoked for the prebiotic synthesis of peptides. Similarly, iron-sulfur clusters are often cited as an example of an ancient catalyst that helped prune early chemical systems into metabolic-like pathways. Because extant iron-sulfur clusters are metallocofactors of protein enzymes and nearly ubiquitous across biology, a reasonable hypothesis is that prebiotic iron-sulfur peptides formed on the early Earth. However, iron-sulfur clusters are coordinated by multiple cysteine residues, and the stability of cysteines to the heat steps of wet-dry cycles has not been determined. It, therefore, has remained unclear if the peptides needed to stabilize the formation of iron-sulfur clusters could have formed. If not, then iron-sulfur-dependent activity may have emerged later, when milder, more biological-like peptide synthesis machinery took hold. Here, we report the thermal stability of cysteine-containing peptides. We show that temperatures of 150 °C lead to the rapid degradation of cysteinyl peptides. However, the presence of Mg2+ at environmentally reasonable concentrations leads to significant protection. Thiophilic metal ions also protect against degradation at 150 °C but require concentrations not frequently observed in the environment. Nevertheless, cysteine-containing peptides are stable at lower, prebiotically plausible temperatures in seawater, carbonate lake, and ferrous lake conditions. The data are consistent with the persistence of cysteine-containing peptides on the early Earth in environments rich in metal ions. High concentrations of Mg2+ are common intra- and extra-cellularly, suggesting that the protection afforded by Mg2+ may reflect conditions that were present on the prebiotic Earth.

Pedal to the Metal: The Homogeneous Catalysis of the Native Chemical Ligation Reaction.

The native chemical ligation reaction of peptide thioesters with cysteinyl peptides is a pivotal chemical process in the production of native or modified peptides and proteins, and well beyond in the preparation of various biomolecule analogs and materials. To benefit from this reaction at its fullest and to access all the possible applications, the experimentalist needs to know the factors affecting its rate and how to control it. This concept article presents the fundamental principles underlying the rate of the native chemical ligation and its homogeneous catalysis by nucleophiles. It has been prepared to serve as a quick guide in the search for an appropriate catalyst.

Flavin catalyzed desulfurization of peptides and proteins in aqueous media.

A biomimetic method has been established for the chemo-selective desulfurization of cysteinyl peptides and proteins in aqueous media. The derivatives of biocatalytic cofactors, flavins, were found to be efficient photosensitizers in a thiyl-radical-mediated desulfurization of Cys. The reaction was conducted in an ultrafast manner with both polypeptides and proteins.

Chemoselective Disulfide Formation by Thiol-Disulfide Interchange in SIT-Protected Cysteinyl Peptides.

Chemoselective disulfide formation is accomplished through a thiol-disulfide interchange approach using sec-isoamyl mercaptan (SIT) as an alkyl sulfenyl-protecting group of one of the Cys residues involved in the pairing. SIT has a dual and unique characteristic, acting as a masking group during the synthesis and directing disulfide formation in the presence of a free thiol. This novel approach is illustrated by the synthesis of several peptides of biological interest.

A straightforward methodology to overcome solubility challenges for N-terminal cysteinyl peptide segments used in native chemical ligation.

One of the main limitations encountered during the chemical synthesis of proteins through native chemical ligation (NCL) is the limited solubility of some of the peptide segments. The most commonly used solution to overcome this problem is to derivatize the segment with a temporary solubilizing tag. Conveniently, the tag can be introduced on the thioester segment in such a way that it is removed concomitantly with the NCL reaction. We herein describe a generalization of this approach to N-terminal cysteinyl segment counterparts, using a straightforward synthetic approach that can be easily automated from commercially available building blocks, and applied it to a well-known problematic target, SUMO-2.

Efficient Chemical Protein Synthesis using Fmoc‐Masked N‐Terminal Cysteine in Peptide Thioester Segments

AbstractWe report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one‐pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N‐masking group of the N‐terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o‐aminoanilide. The ready availability of Fmoc‐Cys(Trt)‐OH, which is routinely used in Fmoc solid‐phase peptide synthesis, where the Fmoc group is pre‐installed on cysteine residue, minimizes additional steps required for the temporary protection of the N‐terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.

Efficient Chemical Protein Synthesis using Fmoc-Masked N-Terminal Cysteine in Peptide Thioester Segments.

We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one‐pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N‐masking group of the N‐terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o‐aminoanilide. The ready availability of Fmoc‐Cys(Trt)‐OH, which is routinely used in Fmoc solid‐phase peptide synthesis, where the Fmoc group is pre‐installed on cysteine residue, minimizes additional steps required for the temporary protection of the N‐terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations.

The native chemical ligation reaction (NCL) involves reacting a C-terminal peptide thioester with an N-terminal cysteinyl peptide to produce a native peptide bond between the two fragments. This reaction has considerably extended the size of polypeptides and proteins that can be produced by total synthesis and has also numerous applications in bioconjugation, polymer synthesis, material science, and micro- and nanotechnology research. The aim of the present review is to provide a thorough mechanistic overview of NCL and extended methods. The most relevant properties of peptide thioesters, Cys peptides, and common solvents, reagents, additives, and catalysts used for these ligations are presented. Mechanisms, selectivity and reactivity are, whenever possible, discussed through the insights of computational and physical chemistry studies. The inherent limitations of NCL are discussed with insights from the mechanistic standpoint. This review also presents a palette of O, S-, N, S-, or N, Se-acyl shift systems as thioester or selenoester surrogates and discusses the special molecular features that govern reactivity in each case. Finally, the various thiol-based auxiliaries and thiol or selenol amino acid surrogates that have been developed so far are discussed with a special focus on the mechanism of long-range N, S-acyl migrations and selective dechalcogenation reactions.

Resin-Bound Crypto-Thioester for Native Chemical Ligation.

The resin-bound N-sulfanylethylanilide (SEAlide) peptide was found to function as a crypto-thioester peptide. Exposure of the peptide resin to an aqueous solution under neutral conditions in the presence of thiols affords thioesters without accompanying racemization of C-terminal amino acids. Furthermore, the resin-bound SEAlide peptides react with N-terminal cysteinyl peptides in the absence of phosphate salts to afford ligated products, whereas soluble SEAlide peptides do not. This unexpected difference in reactivity of the SEAlide peptides allows for a one-pot/three-fragment ligation using resin-bound and unbound peptides.

1,2,4-Triazole-aided native chemical ligation between peptide-N-acyl-N'-methyl-benzimidazolinone and cysteinyl peptide.

1,2,4-Triazole facilitated native chemical ligation (NCL) between peptide-MeNbz (MeNbz: N-acyl-N'-methyl-benzimidazolinone) and a cysteinyl peptide in the absence of thiol additives. The method enabled one-pot desulfurization and iodine oxidation after NCL. Additionally, the direct isolation of the target peptide from the NCL reaction mixture with an activated thiopropyl-Sepharose resin was achieved.

Kinetically Controlled Chemoselective Cyclization Simplifies the Access to Cyclic and Branched Peptides.

A bis(2-sulfanylethyl)amido group reacts significantly faster with cysteinyl peptides when installed on the C-terminal end of a peptide in comparison with the side-chain of Asp and Glu. This property enabled the design of a kinetically controlled chemoselective peptide cyclization reaction, giving straightforward access to cyclic and branched peptides in one pot.

Visible-Light-Induced Specific Desulfurization of Cysteinyl Peptide and Glycopeptide in Aqueous Solution.

Visible-light-induced specific desulfurization of cysteinyl peptides has been explored. The photocatalytic desulfurization catalyzed by Ru(bpy)3(2+) can proceed efficiently at room temperature in aqueous solution or in binary mixtures of aqueous/organic solvent and be compatible with the presence of residues of amino acids, carbohydrates, and various sulfur-containing functional groups. This approach was successfully applied to synthesize linear and cyclic peptides through the ligation-desulfurization protocol.

MTRAQ-based quantitative analysis combined with peptide fractionation based on cysteinyl peptide enrichment

In the present study, the fractionation scheme for cysteinyl peptide enrichment (CPE) was combined with the mass differential tags for relative and absolute quantification (mTRAQ) method to reduce sample complexity and increase proteome coverage. Cysteine residues of the proteins were first alkylated using iodoacetyl PEG2–biotin instead of other conventional alkylating agents such as iodoacetamide. After trypsin digestion, amine groups were labeled with mTRAQ, and these labeled peptides were fractionated according to the presence or absence of cysteine residues using avidin–biotin affinity chromatography. With these approaches, we were able to divide the peptides into the two fractions with more than 90% fractionation efficiency for standard protein and MCF7 cell lysate. When the fractionation strategy was applied to colorectal cancer tissue samples, we were able to obtain quantitative information that was consistent with the previous study based on mTRAQ quantification, implying that the cysteine-based fractionation method does not affect mTRAQ quantification. We expect that the mTRAQ-based quantitative analysis combined with peptide fractionation through the CPE strategy would allow for deep-down analysis of proteome samples and ultimately for increasing proteome coverage with simultaneous quantification for biomarker discovery.

Quantifying reversible oxidation of protein thiols in photosynthetic organisms.

Photosynthetic organisms use dynamic post-translational modifications to survive and adapt, which include reversible oxidative modifications of protein thiols that regulate protein structure, function, and activity. Efforts to quantify thiol modifications on a global scale have relied upon peptide derivatization, typically using isobaric tags such as TMT, ICAT, or iTRAQ that are more expensive, less accurate, and provide less proteome coverage than label-free approaches--suggesting the need for improved experimental designs for studies requiring maximal coverage and precision. Herein, we present the coverage and precision of resin-assisted thiol enrichment coupled to label-free quantitation for the characterization of reversible oxidative modifications on protein thiols. Using C. reinhardtii and Arabidopsis as model systems for algae and plants, we quantified 3662 and 1641 unique cysteinyl peptides, respectively, with median coefficient of variation (CV) of 13% and 16%. Further, our method is extendable for the detection of protein abundance changes and stoichiometries of cysteine oxidation. Finally, we demonstrate proof-of-principle for our method, and reveal that exogenous hydrogen peroxide treatment regulates the C. reinhardtii redox proteome by increasing or decreasing the level of oxidation of 501 or 67 peptides, respectively. As protein activity and function is controlled by oxidative modifications on protein thiols, resin-assisted thiol enrichment coupled to label-free quantitation can reveal how intracellular and environmental stimuli affect plant survival and fitness through oxidative stress.

Selenopeptide transamidation and metathesis.

Selenopeptides can be transamidated by cysteinyl peptides in water using mild conditions (pH 5.5, 37 °C) in the presence of an arylthiol catalyst. Similar conditions also catalyze the metathesis of selenopeptides. The usefulness of the selenophosphine derived from TCEP (TCEP=Se) for inhibiting the TCEP-induced deselenization of selenocysteine residue is also reported.