Cysteine Residues In Peptides Research Articles

Generation of mass tags by the inherent electrochemistry of electrospray for protein mass spectrometry

We present herein a review of our work on the on-line electrochemical generation of mass tags toward cysteine residues in peptides and proteins. Taking advantage of the inherent electrochemical nature of electrospray generated from a microfabricated microspray emitter, selective probes for cysteine were developed and tested for on-line nonquantitative mass tagging of peptides and proteins. The nonquantitative aspect of the covalent tagging thus allows direct counting of free cysteines in the mass spectrum of a biomolecule through additional adduct peaks. Several substituted hydroquinones were investigated in terms of electrochemical properties, and their usefulness for on-line mass tagging during microspray experiments were assessed with L-cysteine, peptides, and intact proteins. Complementarily, numerical simulations were performed to properly understand the respective roles of mass transport, kinetics of electrochemical-chemical reactions, and design of the microspray emitter in the mass tagging overall efficiency. Finally, the on-line electrochemical tagging of cysteine residues was applied to the analysis of tryptic peptides of purified model proteins for protein identification through peptide mass fingerprinting.

Marfey?s reagent for chiral amino acid analysis: A review

The present paper describes characteristics and application of Marfey's reagent (MR) including general protocols for synthesis of the reagent and diastereomers along with advantages, disadvantages and the required precautions. Applications, and comparison with other derivatizing agents, for the resolution of complex mixtures of DL-amino acids, amines and non-proteinogenic amino acids, peptides/amino acids from microorganisms, cysteine residues in peptides, and evaluation of racemizing characteristics have been discussed. Separation mechanisms of resolution of amino acid diastereomers and replacement of Ala-NH2 by suitable chiral moieties providing structural analogs and different chiral variants and their application as a derivatizing agent to examine the efficiency, and reactivity of the reagent have been focussed. Use of MR for preparing CSPs for direct enantiomeric resolution has also been included.

The sulfinic acid switch in proteins.

Recent studies on the redox behaviour of cysteine residues in peptides and proteins have dramatically changed our perspective of the amino acid's role in biocatalysis, intracellular redox sensing and cell signalling. Cysteine sulfinic acid formation in proteins, for example, has long been viewed as an irreversible 'overoxidation' process that might lead to loss of activity, especially under conditions of oxidative stress. Within the last year, several research groups have independently shown that sulfinic acids can be reduced to thiols in vivo. An enzyme with sulfinic acid reductase activity, called sulfiredoxin, has been isolated from yeast and a gene encoding a human analogue has been identified in the human genome. Reversibility of sulfinic acid formation opens the door to a range of yet unexplored redox cycles, cell signalling processes and reduction mechanisms. These cysteine-based redox processes will be of enormous interest to chemists, biochemists, biologists and the medical community alike.

Configuration and racemization determination of cysteine residues in peptides by chiral derivatization and HPLC: application to oxytocin peptides.

An improved RP-HPLC method was developed for the determination of the configuration and stereochemical purity of cysteine residues in peptides. The method consists of oxidation of cysteine and cystine residues to cysteic acid, followed by hydrolysis and pre-column chiral derivatization with Val-Marfey's reagent.

Processing and reactivity of T cell epitopes containing two cysteine residues from hen egg-white lysozyme (HEL74-90).

The Ag processing and structural requirements involved in the generation of a major T cell epitope from the hen egg-white lysozyme protein (HEL74-88), containing two cysteine residues at positions 76 and 80, were investigated. Several T cell hybridomas derived from both low responder (I-Ab) and high responder (I-Ak) mice recognize this region. These hybridomas are strongly responsive to native HEL, but unresponsive to the reduced and carboxymethylated protein. Air-oxidized HEL74-88 peptide was unable to bind I-Ak molecules and failed to stimulate T cells in the absence of intracellular Ag processing. Further functional competition assays showed that alkylation of cysteine residues with bulky methyl groups interferes with the contacts for the MHC class II molecules (I-Ak) of high responder mice and the I-Ab-restricted TCR of low responder mice. Serine substitutions of the cysteine residues of HEL74-88 either enhanced or abrogated T cell stimulation by the peptides without significant alterations in the class II binding. These results suggest that the cysteine residues of peptides must be free from disulfide bonding for efficient stimulation of T cells and yet frequently used modifications of cysteine residues may not be suitable for peptide-based vaccine development.

In vitro depalmitoylation of neurospecific peptides: Implication for infantile neuronal ceroid lipofuscinosis

Palmitoyl protein thioesterase 1 (PPT1) removes palmitate from specific cysteine residues in peptides and proteins. We have previously shown that a palmitoylated myelin glycoprotein. Po octapeptide (IRYCWLRR) can be specifically depalmitoylated by PPT1 in vitro (Cho and Dawson [1998] J. Neurochem. 171;323–329). To characterize further the substrate specificity of PPT1, we prepared various palmitoylated oligopeptides, based on palmitoylated sequences from different proteins. A truncated tetrapeptide from Po (RY[palmitoyl]-CW) was as good a substrate as the octapeptide Po, with optimal activity at pH 4.0. In contrast, other peptide substrates showed marked differences. Thus, the deacylation of GAP-43 (MLCCMRR), rhodopsin (VTTLCCGKN), and Gα subunit (MGCLGNSK) peptides was more efficient at neutral pH (7.4) than at acidic pH (4.0), with the greatest efficiency toward the Gα peptide (five- to sixfold higher than other substrates). Infantile neuronal ceroid lipofuscinosis (INCL) is caused by PPT1 deficiency, and the absence of enzymatic activity was confirmed with GAP-43 peptide as well as the Po peptide. LA-N-5 human neuroblastoma cells overexpressing PPT1 showed increased depalmitoylation of all the peptide substrates, indicating that these peptides are deacylated by PPT1. An amide derivative of a palmitoylated K-Ras peptide (AcG-palmitoyl diamino propionate-VKIKK) acted as an enzyme pseudosubstrate and inhibited PPT1 enzyme activity in a dose-dependent manner. The peptide itself (AcGCVKIKK) did not affect PPT activity. In summary, PPT1 is able to hydrolyze a range of cysteinyl peptide sequences found in both neuron-specific and ubiquitous (e.g., Gα) proteins. The inhibitor of PPT1 activity should facilitate the development of a model for INCL and help explain the neuronal death in this disease. J. Neurosci. Res. 59:32–38, 2000 © 2000 Wiley-Liss, Inc.

In vitro depalmitoylation of neurospecific peptides: Implication for infantile neuronal ceroid lipofuscinosis

Palmitoyl protein thioesterase 1 (PPT1) removes palmitate from specific cysteine residues in peptides and proteins. We have previously shown that a palmitoylated myelin glycoprotein. Po octapeptide (IRYCWLRR) can be specifically depalmitoylated by PPT1 in vitro (Cho and Dawson [1998] J. Neurochem. 171 ;323-329). To characterize further the substrate specificity of PPT1, we prepared various palmitoylated oligopeptides, based on palmitoylated sequences from different proteins. A truncated tetrapeptide from Po (RY[palmitoyl]-CW) was as good a substrate as the octapeptide Po, with optimal activity at pH 4.0. In contrast, other peptide substrates showed marked differences. Thus, the deacylation of GAP-43 (MLCCMRR), rhodopsin (VTTLCCGKN), and Galpha subunit (MGCLGNSK) peptides was more efficient at neutral pH (7.4) than at acidic pH (4.0), with the greatest efficiency toward the Galpha peptide (five- to sixfold higher than other substrates). Infantile neuronal ceroid lipofuscinosis (INCL) is caused by PPT1 deficiency, and the absence of enzymatic activity was confirmed with GAP-43 peptide as well as the Po peptide. LA-N-5 human neuroblastoma cells overexpressing PPT1 showed increased depalmitoylation of all the peptide substrates, indicating that these peptides are deacylated by PPT1. An amide derivative of a palmitoylated K-Ras peptide (AcG-palmitoyl diamino propionate-VKIKK) acted as an enzyme pseudosubstrate and inhibited PPT1 enzyme activity in a dose-dependent manner. The peptide itself (AcGCVKIKK) did not affect PPT activity. In summary, PPT1 is able to hydrolyze a range of cysteinyl peptide sequences found in both neuron-specific and ubiquitous (e.g., Galpha) proteins. The inhibitor of PPT1 activity should facilitate the development of a model for INCL and help explain the neuronal death in this disease.

Fragmentation of protonated thioether conjugates of acrolein using low collision energies

The protonated mercapturic acid conjugate of acrolein, S-(3-oxopropyl)-N-acetyl-L-cysteine (I), undergoes facile retro-Michael loss of acrolein in the gas phase. To determine whether extensive loss of acrolein would impede structural characterization of acrolein-peptide adducts, fragmentation reactions of a series of conjugates, formed by 1,4-Michael addition of acrolein to peptides and cysteine derivatives, were investigated at collision cell potentials up to −50 V using a triple quadrupole mass spectrometer. Differences in fragmentation dynamics suggest protonation at the sulfur of the N-acetylcysteine conjugate I facilitates retro-Michael elimination of acrolein with a low activation energy relative to other fragmentations. Analogous fragmentation was eliminated after borohydride reduction of the aldehyde to an alcohol. Retro-Michael fragmentation was not significant for acrolein conjugates of glutathione derivatives, suggesting that proton sequestration occurs in peptides with multiple amide linkages even when the peptide does not contain a basic amino group. An unexpected outcome of these experiments was the observation of a facile gas-phase cleavage of peptides on the N-terminal side of S-(3-oxopropyl)cysteine residues. Such fragmentation behavior may prove useful for locating cysteine residues in peptides.

Analysis of cysteine residues in peptides and proteins alkylated with volatile reagents

Conditions are described for the reduction and alkylation of cysteines in peptides and proteins with volatile reagents by use of triethylphosphine as reductant, bromopropane as alkylating reagent and triethylamine as base. Alkylated samples need only be vacuum dried prior to subsequent analysis steps. Alkylated samples have been acid hydrolyzed and analyzed on an amino acid analyzer with recoveries of cysteine within 10% of the expected value. Alkylated samples have been directly applied to a sequencer membrane, dried on the surface and cysteines identified by sequence analysis without additional wash steps. In addition proteins blotted onto PVDF have been alkylatedin situ and sequenced with identification of cysteines. On the analyzer and sequencer the S-propylcysteine derivative elutes at a unique position allowing for the unambiguous identification of cysteine. Cysteine residues are quantitativly alkylated under the conditions developed. The ease of this procedure allows the routine analysis of cysteine in peptides and proteins without additional, time consuming repurification or dialysis steps.

Quantitative assessment of cysteine and cystine in peptides and proteins following organomercurial derivatization and analysis by matrix-assisted laser desorption ionization mass spectrometry

Protocols for the analysis of the sulfhydryl content in peptides and proteins using chemical derivatization by organomercurial reagents and analysis by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) have been developed. The number of reactive cysteine residues in peptides and proteins can be determined by exploiting the affinity and selectivity of organomercurial reagents for macromolecular thiols. Mass shifts observed in MALDI mass spectra obtained before and after cysteine derivatization with p-hydroxy-mercuribenzoate (pHMB) permit the number of free sulfhydryl groups to be determined. The pHMB derivative of each free cysteine residue provides a mass shift of 321 u, overcoming limitations in the mass resolution of MALDI time-of-flight mass spectrometry. Reactive cysteine residues in a macromolecule can be selectively derivatized by using a fivefold molar excess of pHMB reagent. Total sulfhydryl content (i.e., cysteine and cystine) can be determined after disulfide reduction. However, analyses for total cysteine content are more complex, requiring protein denaturation, cystine reduction, and sample purification before derivatization and analysis by MALDI-MS. Conditions for sample denaturation, alkyl-phosphine reduction, pHMB derivatization, and sample purification by analyte adsorption and desalting on protein transfer membranes, are described for cysteine/cystine analysis performed on microgram (10–200 pmol) quantities of somatostatin, insulin, hemoglobin, and β-lactoglobulin.

Production of Human PTH(1-34) via a Recombinant DNA Technique

The production of hPTH(1-34) by site specific chemical cleavage of [Cys 35]hPTH(1-84) obtained by a biotechnology technique is described. The amino-peptide bond of S-cyanylated cysteine residues in peptides or proteins is specifically cleaved by exposure to an alkaline solution (Stark, G. R. (1977) Methods Enzymol, 47, 129-132). However, when applying this method to [Cys 35]hPTH(1-84), we observed many by-products. Formation of these by-products was suppressed using a short reaction in 0.03N NaOH containing 6M urea at low temperature, and hPTH(1-34) was specifically produced. The product was indistinguishable from standard hPTH(1-34) with respect to chemical and biological characterization.

Photochemically induced nuclear polarization probes for thiol groups in peptides and proteins

4-Hydroxyphenyl substituted protein labelling reagents have been developed as photo-CIDNP probes for accessible SH groups of cysteine residues in peptides and proteins.

S-methylation of cysteine residues in peptides and proteins with dimethylsulfate

A simple, rapid, and quantitative procedure for the protection of reduced SS bonds in proteins by selective methylation with dimethylsulfate is described. The formed S-methylcysteine is a stable derivative well suited for ion-exchange chromatographic (iec) or gas-liquid chromatographic (glc) analysis. Its methyl- or phenyl-thiohydantoin is easily analyzed by gas-liquid chromatography after trimethylsilylation or by thin-layer chromatography (tlc).