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.     

Sensitivity enhancement of CE and CE-MS for the analysis of peptides by a dynamic pH junction

An analytical method of CE-MS and CE with an online preconcentration technique induced by a dynamic pH junction, addition of organic solvent and large volume injection was developed for sensitive determination of peptides in biological samples. Leucine enkephalin, methionine enkephalin, dynorphin A, β-endorphin and angiotensin II were used as model peptides. The optimal online preconcentration conditions were obtained at a sample matrix consisting of 100?mM borate buffer (pH 10.0) with 50% v/v acetonitrile and a BGE containing 1?M formic acid at pH 2.0, along with a 25-cm injection length. Under the optimized conditions, a 4.0×10(3)-1.1×10(4)-fold increase in peak intensity was achieved without degrading the peak shape. This online preconcentration method was applied to analyze the intracellular angiotensin II within the peptides extracted from HL1 cells and approximately increase of 1×10(4)-fold sensitivity was achieved compared to normal condition. Thus, the developed method could be applied to the analysis of various peptides for peptidomics study in biological samples.     

High-performance liquid chromatographic determination of peptides released by tryptic degradation from opioid peptide precursors in rat brain.

A high-performance liquid chromatographic method involving postcolumn fluorescence derivatization is described for the quantification of five fragment peptides (methionine-enkephalin-Thr-Ser-Glu-Lys, methionine-enkephalin-Lys, methionine-enkephalin-Arg, leucine-enkephalin-Lys and leucine-enkephalin-Arg) released by tryptic digestion from the opioid peptide precursors (proopiomeranocortin and proenkephalins A and B) in rat brain tissues. The tissue proteins containing the precursors are hydrolyzed with trypsin to the fragment peptides. The peptides are separated on an Asahipak ODP-50 column and on-line detected fluorometrically by using hydroxylamine, cobalt(II) and borate buffer reagents. The detection limits (S/N = 3) for the peptides are 0.7-2.8 pmol per 100 microliters injected. The distribution of the precursors in the brain tissues was also discussed on the basis of the determined values of the fragment peptides.     

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.     

Multi-dimensional high-performance liquid chromatography of opioid peptides following pre-column derivatization with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide ion. Preliminary results on the determination of leucine- and methionine-enkephalin-like fluorescence in the striatum region of the rat brain

The reversed-phase high-performance liquid chromatography of three synthetic opioid peptides, 5leucine-enkephalin, 5methionine-enkephalin and [D-2alanine]-5methionine enkephalin, has been studied after their pre-column fluorogenic derivatization with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide to the corresponding 1-cyanobenz[f]isoindole (CBI) derivatives. The chromatographic properties of the three synthetic CBI-peptides were characterized using three different stationary phases, ODS Hypersil, CPS Hypersil and Spherisorb Phenyl, eluted with mobile phases containing various concentrations of methanol, tetrahydrofuran or acetonitrile in 26 mM trifluoroacetic acid, adjusted to pH 3.5. The data obtained using single chromatographic columns were used to design a multi-dimensional system in which the three synthetic CBI-peptides of interest were transferred as a single fraction from one column to a second. The first column served to separate the peptides from the majority of the material in the samples, and the second column was used to separate the three CBI-peptides from each other. The best separation was achieved in which the first column was Spherisorb Phenyl and the second column was ODS Hypersil. Both columns were eluted with a mobile phase of 45% acetonitrile (v/v) in 26 mM trifluoroacetic acid (pH 3.5) at a flow-rate of 1.0 ml/min. The method has been applied to the determination of leucine- and methionine-enkephalin-like fluorescence in the striatum of the rat brain.     

Radioreceptor Assay of Opioid Peptides in Selected Canine Brain Regions

Abstract

A radioreceptor assay using the opioid delta receptor-preferring ligand D- ²ala, D- ⁵leu leucine enkephalin (³H-DADL) and the broader-specificity ligand ³H-etorphine was used to measure five HPLC-purified neuropeptide fractions derived from the peptide-rich fraction of tissue homogenates of nine anatomical regions of the canine brain. The receptoractive peptides studied were methionine enkephalin, alpha-neo-endorphin, dynorphin 1-8, methionine enkephalin-Arg-Phe, and leucine enkephalin. These peptides derive from two larger precursors: proenkephalin A, which contains methionine enkephalin, leucine enkephalin, methionine enkephalin-Arg-Phe; and proenkephalin B, which contains alpha-neo-endorphin and dynorphin 1-8. Receptoractive peptides were measured in the peptide-rich fraction derived from homogenates of canine hypothalamus, pituitary, caudate nucleus, amygdala, hippocampus, mid-brain, thalamus, pons-medulla, and cortex.     

脑啡肽的固相合成与LC-MS/MS分离鉴定

多肽在生命过程中扮演着重要的角色,对其生理生化功能的研究与应用,离不开对单一多肽物质的需求,而化学合成法是获取目标多肽的最有效方法之一。对合成产物的分离与鉴定,是优化合成条件,以得到高产率的重要保证。以两种内源性神经肽亮氨酸脑啡肽和甲硫氨酸脑啡肽为模型,利用Fmoc固相多肽合成策略对其进行合成,并建立了HPLC-ESI-MS/MS新方法用于所制备的亮氨酸脑啡肽和甲硫氨酸脑啡肽的分离与结构鉴定。研究结果显示,主要合成产物均为目标多肽,副产物主要包括C端丢失1个氨基酸所形成的四肽,以及由于甲硫氨酸残基氧化而形成的含甲硫氨酸亚砜的多肽。该研究为高效合成含敏感氨基酸的生理活性多肽提供了新信息。     

High-performance liquid chromatographic determination of substance P-like arginine-containing peptide in rat brain by on-line post-column fluorescence derivatization with benzoin

A high-performance liquid chromatographic method with fluorescence detection is described for the determination of substance P, one of the neuropeptides, in the hypothalamus tissue of rat brain. The detection is based on on-line post-column fluorescence derivatization selective for arginine-containing peptides. The endogenous substance P-like arginine-containing peptide extracted from the tissue and [D-Phe11]-neurotensin as an internal standard were separated from various interfering substances on a reversed-phase column (TSKgel ODS-120T) by gradient elution with acetonitrile-phosphate buffer (pH 2.3). The peptides in the eluate were then automatically converted into fluorescent derivatives for detection by reaction with benzoin. Arginine-containing fragments produced by the enzyme reaction of substance P in the chromatographic fraction with trypsin were also detected, for the identification of the endogenous substance P-like arginine-containing peptide. The method was sensitive enough to permit the quantitative determination of the peptide at a concentration as low as 580 fmol/mg of protein in the brain homogenate. The concentration values of the substance P-like arginine-containing peptide in the tissue were 9.45 +/- 1.50 pmol/mg of protein (six determinations).     

Separation of peptides by HPLC using a surface-confined ionic liquid stationary phase

A butylimidazolium bromide surface-confined ionic liquid stationary phase was synthesized in-house. The synthesized phase was investigated for the separation of five peptides (Gly-Tyr, Val-Tyr-Val, leucine enkephalin, methionine enkephalin, and angiotensin-II). The peptides were successfully separated in less than 5 min. The effect of trifluoroacetic acid (TFA) on the separation of peptides was evaluated with results confirming that TFA was not acting as ion-pairing agent in separation of peptides on this phase.     

Determination of enkephalin peptides by nonaqueous capillary electrophoresis with electrochemical detection

Nonaqueous capillary electrophoresis with electrochemical detection (NACE-ED) was applied to the analysis of enkephalin peptides. The effect of different buffer compositions on the electrophoretic behavior of methionine enkephalin, leucine enkephalin, and [D-Ala2]-leucine enkephalin was studied. Separation of the protonated and the deprotonated peptides was obtained using ACN/methanol-based electrolyte systems. The electrochemical behavior of the enkephalins was studied by the capillary batch injection analysis technique. NACE-ED yielded well-defined signals in the oxidation mode only for the negatively charged analytes. The optimized BGE for the counterelectroosmotic separation consisted of 10 mM ammonium acetate in ACN/methanol (3:1 v/v). Using a platinum microdisk electrode set to an actual potential of +0.65 V detection limits in the submicromolar range were observed which are about one order of magnitude lower compared to UV detection. Problems concerning EOF instability and electrode fouling caused by water and other neutral sample impurities transported by the EOF can be avoided in the EOF-inverted mode using poly(ethylene glycol)-coated capillaries and an actual working electrode potential of +1.0 V. For the quantification of the enkephalins [D-Ala2]leucine enkephalin was used as internal standard. The practical utility for the determination of enkephalins in spiked plasma samples after SPE was demonstrated.     

Advantages of derivatization by osmium tetroxide and 2,2'-bipyridine for matrix-assisted laser desorption/ionization post-source decay fragment ion analysis of peptides

Matrix-assisted laser desorption/ionization--post-source decay (MALDI-PSD) fragment ion analysis is frequently used for peptide sequence determination. PSD fragmentation is often changed or improved in terms of, e.g., sequence coverage, after derivatization. In this work, the influence of modification by an osmium tetroxide-bipyridine reagent (Os,bipy) on the MALDI-PSD behaviour of peptides is studied. The reagent modifies peptides specifically at tryptophan residues and oxidizes methionine to methionine sulfone and cysteine to cysteic acid. As a result the masses of some of the fragments are specifically shifted in case of peptides containing a methionine by +32 Da and, in cases of peptides containing a cysteine residue, by +48 Da. In addition, due to the change in protonation properties of a peptide after oxidation, fragments containing cysteic acid are in most cases totally suppressed. This effect significantly facilitates peptide sequence determination. Improvement of MALDI-TOFMS and PSD analysis after the reaction with Os,bipy is demonstrated for examples involving derivatives of humanin, a novel neuroprotective peptide.     

Existence of low-molecular-weight thiols in Caenorhabditis elegans demonstrated by HPLC-fluorescene detection utilizing 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide

A highly sensitive and simple method using HPLC-fluorescence detection with 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) as a fluorogenic reagent demonstrated the existence of the low-molecular-weight thiols in the extract of Caenorhabditis elegans (C. elegans). The method includes derivatization of the thiols with DAABD-Cl at 40 degrees C for 10 min in borate buffer (pH 9.0) containing TCEP, CHAPS and EDTA, separation of the derivatives on an ODS column and fluorometric determination of the derivatives at 510 +/- 15 nm with excitation at 400 +/- 15 nm. The identification of the thiols was made by HPLC-electrospray ionization mass spectrometry (LC-MS) following isolation of the derivatives using HPLC-fluorescence detection. Low-molecular-weight thiols were found to exist in the extract of C. elegans, such as cysteine, cysteinylglycine, gamma-glutamylcysteine, reduced glutathione and two other unidentified thiol compounds, confirming the existence of the 'glutathione cycle' in C. elegans similar to the mammalian body.     

Improved high-performance liquid chromatographic determination of thiamine and its phosphate esters in animal tissues

An improved method for the determination of thiamine and its phosphate esters in animal tissues using reversed-phase high-performance liquid chromatography with precolumn derivatization is described. Thiamine and its phosphate esters were converted into fluorophores by alkaline cyanogen bromide, and the derivatives were applied to an ODS packed column. Then the effluent obtained by an acidic mobile phase was mixed with an alkaline methanol solution to increase the fluorescence intensity of the derivatives which was determined spectrofluorometrically. A complete, rapid and quantitative separation of thiamin and its phosphate esters was achieved and the use of the acidic buffer as a mobile phase improved the column stability. The fluorophores of thiochrome ester peaks on the chromatogram were sensitive to pretreatment with thiamine triphosphatase or acid phosphatase. The applicability of the method to the determination of the form of thiamin in various tissues of rat is demonstrated.     

Sensitive method of detection, quantitation and purification of peptides using pre-column derivatization with phenyl isothiocyanate

A sensitive method for the detection, quantitation and purification of peptides is described. The method is based on pre-column derivatization of peptides with phenyl isothiocyanate to form phenylthiocarbamoyl derivatives (PTC peptides). The derivatized peptides are analysed by reversed-phase high-performance liquid chromatography on a Zorbax ODS column (5 micron) and detected at 269 nm with a sensitivity limit of 1-5 pmol. The technique was utilized for the separation of a mixture of closely related synthetic peptides. The eluted PTC peptides were collected with an average recovery yield of 75% as determined by amino acid analysis. This method of separation of PTC peptides was also combined with the determination of the complete structure of recovered PTC-dynorphin A-(1-13) using the solid-phase sequenator (Sequemat). The advantages of the derivatization method are the rapidity and completeness of the reaction, the stability of the product, the sensitivity and specificity of the detection of derivatized peptides and the compatibility of the technique with subsequent analytical procedures. A particular application of this method was exemplified by the dosage of enkephalins secreted from perfused bovine adrenal glands.     

Purification of hydrophilic and hydrophobic peptide fragments on a single reversed phase high performance liquid chromatographic column

Hydrophilic peptides generated from enzymic fragmentation of proteins are difficult to purify because they are either weakly bound or unretained by the reversed phase C₁₈ columns favoured for liquid chromatographic separation of peptide mixtures. To overcome this difficulty, peptides that were not bound or only weakly bound by a C₁₈ RP column were reacted with phenyl isothiocyanate (PITC), as used in the initial step in Edman sequencing. The hydrophobic phenylthiocarbamyl (PTC) peptide derivatives produced by the reaction were rechromatographed on the same column. Peptides generated by tryptic digestion of equine cytochrome C were used as a model system to test whether a complete set of peptide fragments could be purified by this method using just one column and solvent system. All the expected hydrophobic tryptic peptides bound to the RP column and were resolved by elution with acetonitrile, but no hydrophilic peptides were recovered as pure fractions. The column breakthrough fraction was reacted with PITC and rechromatographed on the same column, producing a profile consisting of 19 bound peaks. Further rechromatography of some of the fractions at different column temperatures enabled all six of the expected hydrophilic peptides to be purified and identified. The technique has also been applied to the sequence determination of coat protein from peanut stripe potyvirus protein, eight hydrophilic tryptic peptides being recovered and identified as PTC derivatives.     

Separation of N1- and N8-acetylspermidine isomers by reversed-phase column liquid chromatography after derivatization with dansyl chloride

The separation of dansyl derivatives of N1- and N8-acetylspermidine by reversed-phase column liquid chromatography is reported. The influence of organic solvents on the retention of acetylspermidines was studied. Best resolutions were achieved using a C18 column and a ternary mobile phase composed of water, methanol and acetonitrile. The precolumn derivatization method permitted the detection of picomole quantities. A method for the determination of acetylspermidines in rat tissues is described.     

Structural characterization of methionine and leucine enkephalins by hydrogen/deuterium exchange and electrospray ionization tandem mass spectrometry

Conformational changes in two endogenous opioid active pentapeptides methionine enkephalin (Met-enk) and leucine enkephalin (Leu-enk) induced by trifluoroethanol (TFE) were identified using hydrogen/deuterium exchange (HDX), coupled with electrospray ionization (ESI) mass spectrometry. The exchange features in individual amino acid residues were characterized by acquiring tandem mass spectra of the deuterated peptides. The exact identity of the labile hydrogens involved in HDX reveals that the monomer forms of both peptides adopt an unfolded conformation in aqueous solvent, but prefer the 5-->2 beta-turn secondary structure under the membrane-mimetic environment. The ESI mass spectra of Met-enk and Leu-enk also reveal that the dimer structure of these peptides coexists with the monomer conformation. The extent of the dimer structure is dependent on the peptide concentration and nature of the solvent. The non-polar solvents facilitate the dimer formation.     

毛细管电泳-激光诱导荧光用于中药制剂中氨基酸成分的分析

建立了一种用于测定中药制剂中氨基酸成分的毛细管电泳-荧光检测方法. 用含有α-环糊精(α-CD)的硼砂缓冲溶液为背景电解质, 经异硫氰酸荧光素(FITC)衍生的5种氨基酸在50 min内可以得到很好的分离和测定. 考查了各个分离参数对分离的影响, 得到的优化条件为: 含45 mmol/L的α-环糊精的80 mmol/L硼砂缓冲溶液(pH值9.2)作为背景电解质, 分离电压20 kV; 柱温22 ℃. 衍生试剂FITC与单个氨基酸的化学计量比为4∶1时, 能够获得稳定荧光强度的氨基酸衍生物. 在优化条件下, 各氨基酸成分在73.5～2900 nmol/L 的浓度范围内呈良好的线性关系(相关系数r2为0.9906～0.9998). 保留时间和峰面积的相对标准偏差分别为0.8%～3.0%和0.7%～5.7%, 检测限(3倍信噪比)为3.5～35 nmol/L. 该方法准确可靠, 可用于质量控制为目的的中药制剂中氨基酸成分的定量测定.     

Selective determination of tryptophan-containing peptides through precolumn derivatization and liquid chromatography using intramolecular fluorescence resonance energy transfer detection.

A selective and sensitive fluorometric determination method for native fluorescent peptides has been developed. This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the amino groups of tryptophan (Trp)-containing peptides. In this detection process, we monitored the FRET from the native fluorescent Trp moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 10 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated FRET most effectively. The OPA derivatives of the native fluorescent peptides emitted OPA fluorescence (445 nm) through an intramolecular FRET process when they were excited at the excitation maximum wavelength of the Trp-containing peptides (280 nm). The generation of FRET was confirmed through comparison with the analysis of a non-fluorescent peptide (C-reactive protein fragment (77 - 82)) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the Trp-containing peptides when performing LC on a reversed-phase column. The detection limits (signal-to-noise ratio = 3) for the Trp-containing peptides, at a 20-microL injection volume, were 41 - 180 fmol. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of the system that takes advantage of the conventional detection of OPA derivatives. Moreover, native non-fluorescent amines and peptides in the sample monitored at FRET detection are weaker than those of conventional fluorescence detection.     

Micro-electrospray mass spectrometry: Ultra-high-sensitivity analysis of peptides and proteins

A “micro-electrospray” ionization source has been developed that markedly increases the sensitivity of the conventional electrospray source. This was achieved by optimization of the source to accommodate nanoliter flow rates from 300 to 800-nL/min spraying directly from a capillary needle that, for the analysis of peptides, contained C18 liquid chromatography packing as an integrated concentration-desalting device. Thus, a total of 1 fmol of methionine enkephalin was desorbed from the capillary column spray needle, loaded as a 10-μL injection of 100-amol/μL solution. The mass spectrum showed the [M + H]⁺ ion at m/z 574.2 with a signal-to-noise ratio of better than 5:1 from a chromatographic peak with a width of about 12 s. A narrow range (15-u) tandem mass spectrum was obtained for methionine enkephalin from the injection of 500 amol, and a full-scan tandem-mass spectrum was obtained from 50 fmol. For proteins, the average mass measurement accuracy was approximately 100–200 ppm for the injection of 2.5 fmol of apomyoglobin and 20–40 ppm for 200 fmol. Carbonic anhydrase B and bovine serum albumin showed similar mass measurement accuracies.