Assay for enkephalin-degrading peptidases in rat brain tissues by high-performance liquid chromatography with on-line post-column fluorescence detection

The activities of enkephalin-degrading peptidases such as enkephalinases A and B in rat brain tissues were simultaneously assayed by a high-performance liquid chromatographic method with fluorimetric detection with an automatic reaction system. Tyrosine and tyrosine-containing peptides produced enzymatically from the substrate, methionine-enkephaline, were separated by gradient elution on a reversed-phase column (TSK gel ODS-120T), and then converted into fluorescent derivatives for detection by reaction with hydroxylamine, cobalt(II) and borate reagents. The method permits the simple and sensitive detection of N-terminal tyrosine-containing fragments of the enkephalin peptide. The limits of detection are 5-20 pmol per assay tube for the N-terminal tyrosine-containing fragments. The enzyme activities in the regionally separated tissues were 54-191 pmol/min.mg protein for enkephalinase A and 79-153 pmol/min.mg protein for enkephalinase B, which were calculated from the formation of Tyr-Gly-Gly and Tyr-Gly, respectively, during the enzyme reaction.     

On-line post-column fluorescence detection for N-terminal tyrosine-containing peptides in high-performance liquid chromatography

A detection system based on on-line post-column fluorescence derivatization is described for the determination of N-terminal tyrosine-containing peptides by reversed-phase high-performance liquid chromatography. The peptides are automatically converted into fluorescent derivatives by reaction with hydroxylamine, cobalt (II) and borate after peptide separation on a reversed-phase column (TSKgel ODS-120T) followed by passage through an ultraviolet absorbance detector. The reaction system permits the fluorescence detection at 435 nm (emission) with excitation at 335 nm for N-terminal tyrosine-containing synthetic peptides in as little as picomole amounts. The facile fluorescence detection of N-terminal tyrosine-containing fragments produced from methionine-enkephalin by enzymatic degradation using a rat brain homogenate was achieved by comparison with the ultraviolet absorption detection at 215 nm.     

Selective determination of N-terminal tyrosine containing peptides by a novel fluorescence reaction with borate,hydroxylamine and cobalt(II)

A fluorimetric method is proposed for determining N-terminal tyrosine-containing peptides, of which some peptides such as enkephalins and kyotorphin are of physiological importance. An intense fluorescence is produced when the peptide is heated at 100°C for 3 min in a weakly alkaline medium containing borate, hydroxylamine and cobalt(II). The fluorescent species is stabilized with β-mercaptoethanol, with excitation and emission maxima at 335 and 430 nm, respectively. The method is highly selective for N-terminal tyrosine-containing peptides, with a detection limit of 43–69 pmol ml^?1.     

Structure-ionization relationship of tyrosine-containing peptides

The arid-base chemistry of tyrosine-containing peptides such as enkephalin, tyrosylglycylglycine, tyrosylglycine and analogous peptides is described. For each peptide and tyrosine derivative, microscopic and macroscopic acid dissociation constants and the thermodynamic parameters for proton dissociations were determined from pH-titrations and ultraviolet absorption spectra. The relative concentrations of various ionic forms for the peptides were calculated from the microscopic constants. The concentration ratio, represented by the tautomeric equilibrium constant (K(t)), showed a definite relationship to structure.     

Tyrosinase-induced cross-linking of tyrosine-containing peptides investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Tyrosinase-induced oxidation of tyrosine is known to lead to melanin by cross-linking of 5,6-dihydroxyindole (DHI) and indole-5,6-quinone intermediates. However, tyrosinase-induced cross-linking of tyrosine-containing peptides has not been reported. We observed tyrosinase-induced adducts of tyrosine-containing peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). MALDI-TOFMS was also used to observe tyrosine adducts at various levels of oxidation derived from acid hydrolysis of the peptide adducts. The rate of tyrosinase-induced browning of lys-tyr-lys was about half of that of tyrosine. These results indicate that tyrosinase-induced browning of tyrosine-containing peptides via direct oxidation and cross-linking of the benzene ring of the tyrosine residue occurs at a significant rate and needs to be considered in melanogenesis.     

Negative ion dissociation of peptides containing hydroxyl side chains

The dissociation of deprotonated peptides containing hydroxyl side chains was studied by electrospray ionization coupled with Fourier transform ion cyclotron resonance (ESI-FTICR) via sustained off-resonance irradiation collision induced dissociation (SORI-CID). Dissociation under post-source decay (PSD) conditions was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). This work included hexapeptides with one residue of serine, threonine, or tyrosine and five inert alanine residues. During SORI-CID and PSD, dissociation of [M-H](-) yielded c- and y-ions. Side-chain losses of formaldehyde (HCHO) from serine-containing peptides, acetaldehyde (CH(3)CHO) from threonine-containing peptides, and 4-methylene-2,5-cycohexadienone (C(7)H(6)O) from tyrosine-containing peptides were generally observed in the negative ion PSD and SORI-CID spectra. Side-chain loss occurs much less from tyrosine-containing peptides than from serine- and threonine-containing peptides. This is probably due to the bulky side chain of tyrosine, resulting in steric hindrance and poor geometry for dissociation reactions. Additionally, a selective cleavage leading to the elimination of the C-terminal residue from [M-H](-) was observed from the peptides with serine and threonine at the C-terminus. This cleavage does not occur in the dissociation of peptides with an amide group at the C-terminus or peptides with neutral or basic residues at the C-terminus. It also does not occur with tyrosine at the C-terminus. Both the C-terminal carboxylic acid group and the hydroxyl side chain of the C-terminal residue must play important roles in the mechanism of C-terminal residue loss. A mechanism involving both the C-terminal carboxylic acid group and a hydroxyl side chain of serine and threonine is proposed.     

Formation of molecular radical cations of enkephalin derivatives via collision-induced dissociation of electrospray-generated copper (II) complex ions of amines and peptides

Fragmentation of some electrospray-generated complex ions, [63CuII(amine)M].2+, where M is an enkephalin derivative, produces the radical cation of the peptide, M.+. This ion has only been observed when M contains a tyrosyl or tryptophanyl residue plus a basic residue, typically arginyl or lysyl. A typical viable amine is diethylenetriamine. Collision-induced dissociation (CID) of the M.+ ion yields a prominent [M - 106].+ product ion for tyrosine-containing peptides, and a prominent [M - 129].+ ion for a tryptophan-containing peptide. These fragment ions are formed as a result of elimination of the tyrosyl and tryptophanyl side chains. Dissociation of these ions, in turn, produces second generation product ions, many of which are typically absent in the fragmentation of protonated peptide ions. Structures for some of these unusual ions are proposed.     

Fluorescence derivatization of bioactive peptides for high-performance liquid chromatography

New fluorescence derivatization techniques are introduced for the quantitative determination of arginine- or tyrosine-containing peptides by high-performance liquid chromatography with fluorescence detection. The methodology offers enhancement of both sensitivity and specificity. It is thus suitable for trace (0.1–10 pmol) analysis of the bioactive peptides such as angiotensins and enkephalins.     

A new approach for sequencing human IRS1 phosphotyrosine-containing peptides using CapLC-Q-TOF(micro)

Reversible phosphorylation of proteins functions as a biological switching network for activation and inhibition of downstream biological processes. Since phosphorylations of these sites are often transient processes, and hence sub-stoichiometric, systematic characterization of phosphorylation sites is a formidable challenge. In this work, a new approach was developed to pinpoint phosphotyrosine sites on tyrosine-containing peptides. This required (1) the development of a new and highly sensitive nano-electrospray assembly and (2) validation of the concept that the specificity and detection limit for trace levels of phosphotyrosine immonium ion in peptide mixtures from protein digests can be increased by varying the collision energy. With our method, an automatic tandem mass spectrometric analysis of peptides eluted from a C(18) capillary liquid chromatographic column is triggered by a positive confirmation of phosphotyrosine immonium ion in a time-of-flight mass spectrometric survey. The approach was tested by analyzing the phosphorylation of human IRS-1 peptides that interact with the Src-homology 2 domain and mixtures of these peptides with tryptic digests of bovine serum albumin and horse heart myoglobin.     

Enhanced detection of sulfo-peptides as onium salts in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A new two-component system, consisting of a matrix and an onium salt as comatrix, is described for detection of sulfo-peptides in the positive mode by matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Binary iodonium salts were superior to quaternary phosphonium salts in terms of suppression of desulfation and salt formation with the carboxyl group. Of the iodonium salts examined, bis(4-tert-butylphenyl)iodonium (BTI) hexafluorophosphate and bromide were most effective in giving intensive molecular ion signals in the form of [M(BTI)+BTI](+). The conditions optimized for O-sulfated tyrosine-containing peptides could be applicable for O-sulfated serine- and threonine-containing peptides. In the case of a phospho-peptide, a molecular ion appeared more intensively as a proton adduct than as a BTI adduct.     

Application of capillary reversed-phase high-performance liquid chromatography to high-sensitivity protein sequence analysis.

A continuous gradient elution method for capillary column (less than 0.32 mm I.D.) liquid chromatography was developed. Gradient eluent from a microbore liquid chromatograph was split ahead of the injector so that an accurate percentage (2-3%) of the mobile phase delivered by the pump flowed through the capillary column. The outlet of the column was connected to a length of 0.075 mm I.D. fused-silica capillary tubing which, in turn, was connected to a 6-mm optical path length longitudinal capillary flow cell. Fused-silica capillary columns of 0.32 mm I.D. were slurry-packed efficiently with 7-microns spherical, 300 A pore size, C8 bonded-phase particles, and evaluated in terms of their ability to resolve mixtures of proteins, peptides or phenylthiohydantoin (PTH)-amino acid derivatives. The gradient elution profiles agreed with those obtained using microbore (less than 2.1 mm I.D.) and larger bore columns. The minimum detectable amounts for proteins and PTH-amino acids on 0.32 mm I.D. capillary columns were 50 pg and 25 fmol, respectively. At a flow-rate of 3.6 microliters/min, proteins and peptides were recovered from the capillary columns in volumes of about 2-8 microliters. The use of a multiple-wavelength, forward-optics detector for identifying tryptophan- and tyrosine-containing peptides is discussed.     

Synthesis of 3,5-difluorotyrosine-containing peptides: application in substrate profiling of protein tyrosine phosphatases

Fully protected 3,5-difluorotyrosine (F2Y), Fmoc-F2Y(tBu)-OH, is efficiently prepared by a chemoenzymatic process and incorporated into individual peptides and combinatorial peptide libraries. The F2Y-containing peptides display kinetic properties toward protein tyrosine phosphatases (PTPs) similar to their corresponding tyrosine-containing counterparts but are resistant to tyrosinase action. These properties make F2Y a useful tyrosine surrogate during peptide library screening for optimal PTP substrates.     

ELECTRONIC EFFECTS ON THE FLUORESCENCE OF TYROSINE IN SMALL PEPTIDES

Abstract— It is shown for a series of tyrosine-derivatives and tyrosine-containing peptides that the amide group in combination with electron-withdrawing substituents quenches the fluorescence of the phenol moiety. The ammonium group has the strongest electron-withdrawing effect and thus the largest influence on the quenching rate. The peptide group itself does not quench the fluorescence. In a series of peptides with an increasing number of alanines the decreasing quenching efficiency or the peptide group due to the greater distance of the ammonium group is demonstrated. In tyrosine-containing di- and tripeptides a linear correlation between the ¹³C-NMR chemical shift δ of the C₂ atom of various aliphatic amino acids and the fluorescence-quenching constant confirms the hypothesis that electron-withdrawing and donating groups are modulating the fluorescence-quenching efficiency of the peptide group. In small peptides the fluorescence lifetime of tyrosine is characteristic for the neighboring amino acids. Using model substances the redox properties of a peptide group and the phenol ring were studied electrochemically. The highest occupied molecular orbital of the tyrosine (1.4 V vs saturated calomel electrode [SCE]) and the lowest unoccupied molecular orbital of the peptide group (-3.12 V vs SCE) have appropriate energies for a photoinduced electron transfer reaction. For solute-quenching experiments quencher molecules can be systematically selected.     

Label-free electrochemical differentiation of phosphorylated and non-phosphorylated peptide by electro-catalyzed tyrosine oxidation

Protein phosphorylation plays an important role in many significant cellular processes, and has thus gained tremendous interest in the field of proteomics. The electro-active tyrosine residue, as an important receptor of phosphorylation in proteins, exhibits electro-inactivity after being phosphorylated on the hydroxy group of its aromatic ring. In this study, the electrochemical oxidation of tyrosine on indium tin oxide (ITO) electrodes was catalyzed with an electron mediator Os(bpy)(3)(2+) (bpy = 2,2'-bipyridine) and was employed as a signal reporter to differentially detect non-phosphorylated and phosphorylated peptides. A short, tyrosine-containing peptide glu-glu-glu-glu-glu-tyr (EY-6) was immobilized on an ITO surface using the layer-by-layer self-assembly method, and was detected by cyclic voltammetry in an Os(bpy)(3)(2+) solution. The limit of detection was about 0.23 microg mL(-1) EY-6 in solution. The phosphorylated peptide glu-glu-glu-glu-glu-tyr-OP (EY-6P) did not produce an appreciable oxidation current on the electrode. Surface plasmon resonance measurements revealed that the amount of EY-6 and EY-6P adsorbed on the sensor chip surface was 269 and 378 pg mm(-2), respectively. The poly(glu, tyr) (4 : 1) peptide, a protein tyrosine kinase substrate, was also detected by the same approach, with a detection limit of 0.65 microg mL(-1). This new approach offers the possibility of label-free and on-chip detection of protein tyrosine kinase activity.     

Peptide Cyclization Mediated by Metal-Free S-Arylation: S-Protected Cysteine Sulfoxide as an Umpolung of the Cysteine Nucleophile

Covalent linking of side chains provides a method to produce cyclic or stapled peptides that are important in developing peptide-based drugs. A variety of crosslinking formats contribute to fixing the active conformer and prolonging its biological activity under physiological conditions. One format uses the cysteine thiol to participate in crosslinking through nucleophilic thiolate anions or thiyl radicals to form thioether and disulfide bonds. Removal of the S-protection from an S-protected Cys derivative generates the thiol, which functions as a nucleophile. S-Oxidation of a protected Cys allows the formation of a sulfoxide that operates as an umpolung electrophile. Herein, the applicability of S-p-methoxybenzyl Cys sulfoxide (Cys(MBzl)(O)) to the formation of a thioether linkage between tryptophan and Cys has been investigated. The reaction of peptides containing Cys(MBzl)(O) and Trp with trifluoromethanesulfonic acid (TFMSA) or methanesulfonic acid (MSA) in TFA in the presence of guanidine hydrochloride (Gn ⋅ HCl) proceeded to give cyclic or stapled peptides possessing the Cys-Trp thioether linkage. In this reaction, strong acids such as TFMSA or MSA are necessary to activate the sulfoxide. Additionally, Gn ⋅ HCl plays a critical role in producing an electrophilic Cys derivative that combines with the indole by aromatic electrophilic substitution. The findings led us to conclude that the less-electrophilic Cys(MBzl)(O) serves as an acid-activated umpolung of a Cys nucleophile and is useful for S-arylation-mediated peptide cyclization.     

Photodissociation pathways and lifetimes of protonated peptides and their dimers

Photodissociation lifetimes and fragment channels of gas-phase, protonated YA(n) (n = 1,2) peptides and their dimers were measured with 266 nm photons. The protonated monomers were found to have a fast dissociation channel with an exponential lifetime of ~200 ns while the protonated dimers show an additional slow dissociation component with a lifetime of ~2 μs. Laser power dependence measurements enabled us to ascribe the fast channel in the monomer and the slow channel in the dimer to a one-photon process, whereas the fast dimer channel is from a two-photon process. The slow (1 photon) dissociation channel in the dimer was found to result in cleavage of the H-bonds after energy transfer through these H-bonds. In general, the dissociation of these protonated peptides is non-prompt and the decay time was found to increase with the size of the peptides. Quantum RRKM calculations of the microcanonical rate constants also confirmed a statistical nature of the photodissociation processes in the dipeptide monomers and dimers. The classical RRKM expression gives a rate constant as an analytical function of the number of active vibrational modes in the system, estimated separately on the basis of the equipartition theorem. It demonstrates encouraging results in predicting fragmentation lifetimes of protonated peptides. Finally, we present the first experimental evidence for a photo-induced conversion of tyrosine-containing peptides into monocyclic aromatic hydrocarbon along with a formamide molecule both found in space.     

Cα-Cβ chromophore bond dissociation in protonated tyrosine-methionine, methionine-tyrosine, tryptophan-methionine, methionine-tryptophan and their sulfoxide analogs

C(α)-C(β) chromophore bond dissociation in some selected methionine-containing dipeptides induced by UV photons is investigated. In methionine containing dipeptides with tryptophan as the UV chromophore, the tryptophan side chain is ejected either as an ion or as a neutral fragment while in dipeptides with tyrosine, the tyrosine side chain is lost only as a neutral fragment. Mechanisms responsible for these fragmentations are proposed based on measured branching ratios and fragmentation times, and on the results of DFT/B3-LYP calculations. It appears that the C(α)-C(β) bond cleavage is a non-statistical dissociation for the peptides containing tyrosine, and occurs after internal conversion for those with tryptophan. The proposed mechanisms are governed by the ionization potential of the aromatic side chain compared to that of the rest of the molecule, and by the proton affinity of the aromatic side chain compared to that of the methionine side chain. In tyrosine-containing peptides, the presence of oxygen on sulfur of methionine presumably reduces the ionization potential of the peptide backbone, facilitating the loss of the side chain as a neutral fragment. In tryptophan-containing peptides, the presence of oxygen on methionyl-sulfur expedites the transfer of the proton from the side chain to the sulfoxide, which facilitates the loss of the neutral side chain.     

Assay of protein drug substances present in solution mixtures by fluorescamine derivatization and capillary electrophoresis.

A method is described to enhance the resolution and detection sensitivity of proteins, peptides, and amino acids in capillary electrophoretic analysis of solution mixtures. The method consists of derivatizing the analytes with fluorescamine, which is normally used as a fluorogenic reagent for compounds containing a reactive primary amine functional group, and then using the derivative as an ultraviolet chromophore to enhance detection sensitivity (measured at 280 nm) in capillary electrophoresis. The results demonstrated a significant improvement in the separation and detection sensitivity of the derivatized analytes as compared to their underivatized counterparts. The use of chromophores, such as fluorescamine, in capillary electrophoresis facilitates the analysis of components of solution mixtures, such as pharmaceutical formulations, that could not be resolved and/or detected by conventional capillary electrophoresis procedures.     

Direct entry to peptidyl ketones via SmI2-mediated C-C bond formation with readily accessible N-peptidyl oxazolidinones

In this work, a new method for the preparation of peptidyl ketones is presented employing a SmI(2)/H(2)O-mediated coupling of N-peptidyl oxazolidinones with electron-deficient alkenes. The requisite peptide imides were easily prepared by solution-phase peptide synthesis starting from an N-acyl oxazolidinone derivative of an amino acid. Importantly, they could be used directly in the C-C bond-forming step without the need for further functionalization. Coupling of these peptide derivatives with a second peptide possessing an N-terminal acryloyl group leads to ketomethylene isosteres of glycine-containing peptides. This method represents an alternative means for ligating two small peptides through a C-C bond-forming step.     

The unusually high proton affinity of aza-18-crown-6 ether: implications for the molecular recognition of lysine in peptides by lariat crown ethers

Recent studies have shown that 18-crown-6 ether (18C6) will selectively form adducts in the gas phase with small, lysine containing peptides. The present study extends this work by investigating the ability of aza-18-crown-6 ether (A18C6) and L1 (a simple lariat crown ether derivative of A18C6) to form similar noncovalent adducts with the side chain of lysine in model peptides in the gas phase. The substitution of nitrogen for oxygen greatly increases the proton affinity of A18C6 relative to 18C6 and inhibits the formation of noncovalent adducts with small lysine containing peptides. The proton affinity of A18C6 is determined by the kinetic method to be 250 +/- 1 kcal/mol. This value is much higher than that for diethanolamine (228 kcal/mol) or for 18C6 (231 kcal/mol). This unusually high basicity is rationalized by semi-empirical calculations that suggest a highly symmetrical structure for protonated A18C6 in which the three most distant oxygens are able to fold back and hydrogen bond with the protonated nitrogen. In the case of L1, the lariat side chain is attached by an amide bond, lowering the proton affinity of LI relative to that of A18C6. This allows L1 to form noncovalent adducts with lysine despite the fact that steric repulsion within the cavity of the crown is increased to some extent. The relative ammonium ion affinities of these various crown ethers are shown to serve as qualitative predictors for the molecular recognition of lysine. The order of the relative ammonium ion affinities is 18C6>L1>A18C6 as determined by the kinetic method. These results suggest that the substitution of nitrogen for oxygen in the crown ether is not beneficial for the molecular recognition of lysine.