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.     

Analysis of the performance of antibody capture methods using fluorescent peptides with capillary zone electrophoresis with laser-induced fluorescence

A method to analyze the performance of an antibody capture method using fluorescent peptides by capillary zone electrophoresis using laser-induced fluorescence (CZE-LIF) for detection has been developed. Fluorescent peptides from the prion protein were synthesized and the corresponding antibodies were produced in rabbits against these peptides. The antibodies were used to capture the fluorescent peptides. The antibodies were then bound to protein A Sepharose. After elution, the amount of fluorescent peptide that was captured vs. the total amount placed in the assay was evaluated by CZE-LIF. Of the three peptides used in this evaluation, it was found that the recovery was approximately 25-35%. When the abnormal prion protein was prepared from scrapie-infected brain samples from hamsters and a sheep using the previously described extraction method and this method, the amount of abnormal prion protein that was measured in the fluorescence immunoassay correlated with amounts estimated from Western blot. We conclude that this method can be used to detect abnormal prion protein in a tissue sample.     

Comprehensive analysis system using liquid chromatography-mass spectrometry for the biosynthetic study of peptides produced by cyanobacteria

Microcystins are hepatotoxic heptapeptides and general tumor promoters produced by several species of the genera Microcystis, Anabaena, Oscillatoria and Nostoc. They are non-ribosomally synthesized via a mixed polyketide synthase/non-ribosomal peptide synthetase system called microcystin synthetase. We have carried out the detection, isolation and structural determination of non-toxic peptides produced together with microcystins by toxic cyanobacteria, which are classified into several groups on the basis of their structures and some of these non-toxic peptides are also non-ribosomally synthesized as well as microcystins. In the present study, we tried to correlate the secondary metabolic peptides produced by the hepatotoxic cyanobacteria with the corresponding peptide synthetase genes. An analytical method using LC-electroscopy ionization MS and photodiode array detection was developed for the exhaustive screening of cyanobacterial peptides in Japanese strains and it was successfully applied to the peptide fractions extracted from these strains. The established method was advantageous over conventional ones using the usual HPLC and matrix-assisted laser desorption ionization time-of-flight MS, because more structural information could be obtained and it is easier to distinguish microcystins from other peptides using this method. Small amounts of other peptides could also be detected by this method. The established method will contribute to the investigation of the relationship between genes encoding the peptide synthetase and secondary metabolic peptides.     

样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响

人乳内源肽是乳蛋白在乳腺中被降解形成的具有生理功能的肽,是人乳的重要组成部分,研究人乳内源肽对于婴儿健康具有重要的意义.高效液相色谱-串联质谱(LC-MS/MS)联用技术的应用,促使人乳内源肽的研究取得了突破性的进展.人乳中内源肽含量低、干扰组分多,样品制备方法是影响分析结果的关键步骤.为了研究样品制备方法对分析结果的...     

Application of MALDI TOF/TOF mass spectrometry and collision-induced dissociation for the identification of disulfide-bonded peptides

This paper describes a method for the fast identification and composition of disulfide-bonded peptides. A unique fragmentation signature of inter-disulfide-bonded peptides is detected using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry and high-energy collision-induced dissociation (CID). This fragmentation pattern identifies peptides with an interconnected disulfide bond and provides information regarding the composition of the peptides involved in the pairing. The distinctive signature produced using CID is a triplet of ions resulting from the cleavage of the disulfide bond to produce dehydroalanine, cysteine or thiocysteine product ions. This method is not applicable to intra-peptide disulfide bonds, as the cleavage mechanism is not the same and a triplet pattern is not observed. This method has been successfully applied to identifying disulfide-bonded peptides in a number of control digestions, as well as study samples where disulfide bond networks were postulated and/or unknown.     

Simultaneous analysis by capillary electrophoresis of five amyloid peptides as potential biomarkers of Alzheimer's disease

We report here a CE method for the separation and quantitation of five amyloid peptides (Abeta1-42, 1-40, 1-39, 1-38, and 1-37) considered as potential biomarkers of Alzheimer's disease. These amyloid peptides have very similar structures. Sample preparation and storage conditions are critical parameters to ensure their solubility and to avoid the aggregation process in particular for Abeta1-42. Their solubility was found fully dependent on the NH(4)OH concentration that was employed initially to dissolve the lyophilized amyloid peptides. Conditions to achieve a full separation of these peptides were found using a dynamic coating with 1,4-diaminobutane (DAB). The linear decrease of their electrophoretic mobility highlighted an ion-pairing phenomenon between the peptides and DAB. The optimal background electrolyte was a 40 mM borate buffer, pH 9 containing 3 mM of DAB. Under these conditions, resolutions ranged from 1.3 to 2.4 with theoretical plates reaching 300,000. Under the retained conditions, we showed that adsorption of peptides to silica was negligible (recovery over 94.5%) and depletion effect of the background electrolyte was overcome. The method was finally validated in terms of linearity and repeatability and the limits of detection for the five Abeta peptides were estimated. The inter-day repeatability of the migration times was very satisfactory with RSDs less than 1.55%. The RSDs of the peak areas were below 5%. With this CE-UV method, limits of detection of the peptides ranged from 300 to 500 nM. We finally demonstrated that this method can be applied to real biological samples such as CSF.     

Development of a capillary electrophoresis method for the characterization of collagens in cartilage tissue

The use of capillary electrophoresis (CE) for the separation of peptides specific to type I and type II collagen is evaluated. The aim of this work is to develop a method to characterize cartilage, cartilage repair tissue, and tissue engineered cartilage. The analysis is dependent on the cleavage of collagen into constituent peptides by cyanogen bromide. A number of these peptides are specific to the collagen type. CE is evaluated for the separation of these specific peptides using uncoated and coated capillaries over a wide range of pH and buffer concentrations. Separation of peptides specific to type I and type II collagen is achieved using a Supelco CElect N capillary and a 100mM phosphate buffer at pH 6. Meniscal cartilage is characterized using this method. The proportion of type I collagen to type II collagen corresponds well with that reported by others and indicates the potential of this method for the characterization of cartilage.     

Reversed approach to S-farnesylation and S-palmitoylation: application to an efficient synthesis of the C-terminus of lipidated human N-ras hexapeptide

[reaction: see text] A general reversed approach is described to synthesize S-palmitoylated and S-farnesylated peptides via S(N)2 displacement of bromide by reaction of a thiol group containing lipid as nucleophile with bromoalanine-containing peptides as electrophile. By employing this approach, lipidated peptides, including characteristic partial structures of human Ras peptides, were synthesized in good yields. This method gives access to farnesylated, palmitoylated, and doubly lipidated peptides.     

High-throughput sequence determination of cyclic peptide library members by partial Edman degradation/mass spectrometry

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. Large combinatorial libraries of cyclic peptides can now be routinely synthesized by the split-and-pool method and screened against biological targets. However, post-screening sequence determination of hit peptides has been problematic. In this report, a high-throughput method for the sequence determination of cyclic peptide library members has been developed. TentaGel microbeads (90 mum) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear peptide as the encoding sequence. After screening of the cyclic peptide library against a macromolecular target, the identity of hit peptides was determined by sequencing the linear encoding peptides inside the bead using a partial Edman degradation/mass spectrometry method. On-bead screening of an octapeptide library (theoretical diversity of 160 000) identified cyclic peptides that bind to streptavidin. A 400-member library of tyrocidine A analogues was synthesized on TentaGel macrobeads and solution-phase screening of the library directly against bacterial cells identified a tyrocidine analogue of improved antibacterial activity. Our results demonstrate that the new method for cyclic peptide sequence determination is reliable, operationally simple, rapid, and inexpensive and should greatly expand the utility of cyclic peptides in biomedical research.     

New, simple method for synthesis of cystine peptides

A report is given on the conversion of S-trityl-cysteine-containing protected peptides to cystine peptides by a reaction with iodine in methanol. The new method permits the synthesis of symmetrical, open-chain unsymmetrical, and especially cyclic cystine peptides.     

Rapid and sensitive identification of epitope-containing peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads

A method has been developed for rapid and sensitive identification of epitope-containing peptides, based on direct MALDI-MS/MS analysis of epitope-containing peptides affinity bound to affinity beads. This technique provides sequence information of the epitope that allows unambiguous identification of the epitope either by database searching or de novo sequencing. With MALDI-MS, affinity beads with bound peptides can be placed directly on the MALDI target and analyzed. Coupling a MALDI source to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer allows direct sequencing of the bound peptides. In contrast to ESI-MS/MS, elution of the affinity-bound peptides followed by additional concentration and purification steps is not required, thus reducing the potential for sample loss. Direct mass spectrometric sequencing of affinity-bound peptides eliminates the need for chemical or enzymatic sequencing. Other advantages of this direct MALDI-MS/MS analysis of epitope-containing peptides bound to the affinity beads include its sensitivity (femtomole levels) and speed. In addition, direct analysis of peptides on affinity beads does not adversely affect the high mass accuracy of a QqTOF, and database searching can be performed on the MS/MS spectra obtained. In proof-of-principle experiments, this method has been demonstrated on beads containing immobilized antibodies against phosphotyrosine, the c-myc epitope tag, as well as immobilized avidin. Furthermore, de novo sequencing of epitope-containing peptides is demonstrated. The first application of this method was with anti-FLAG-tag affinity beads, where direct MALDI MS/MS was used to determine an unexpected enzymatic cleavage site on a growth factor protein.     

Solid-phase capture and release of arginine peptides by selective tagging and boronate affinity chromatography

A method for the selection of arginine-containing peptides from a mixture by a solid phase capture and release technique is presented. The method is based on the covalent modification of the guanidine group of arginine with 2,3-butanedione and phenylboronic acid under alkaline conditions. Using polymeric materials with immobilised phenylboronic acid the arginine-peptides can be captured on a solid support while arginine-free peptides are not covalently bound and can be washed away. Finally, the arginine-peptides can be cleaved again from the boronic acid beads due to the reversibility of the reaction. The recovered peptides are then analysed by liquid chromatography-tandem mass spectrometry. The method was optimised with model peptides with regard to the non-specific binding of arginine-free peptides and quantitative cleavage of the label after the selection step. Using an adequate protocol, the applicability towards more complex samples was successfully tested with a tryptic digest of a mixture of three standard proteins.     

QDs标记免疫调节肽及其与T细胞作用的表征

量子点是直径为1~10 nm的球形半导体纳米晶体, 也被称为半导体量子点, 简称QDs. 与有机荧光染料相比, QDs具有激发光谱单一、 荧光谱线窄、 发光效率高、 发光颜色可调、 可进行多色联合标记, 并且光稳定性好等优点, 所以量子点是非常有前途的生物标记物[^1,2]. 研究结果表明, 量子点可以与许多生物分子如蛋白质、多肽、核酸及小分子配体等偶联. 现已有许多关于量子点标记生物分子的报道, 如用量子点标记木瓜蛋白酶、 胰蛋白酶、 天花粉蛋白和表皮生长因子等^[3-5].用量子点标记生物分子作为荧光探针已成功地应用于多种生物分析, 如DNA杂交监测、 免疫分析和用QDs检测ATP推动的反应等^[4,6,7]. 目前, 对量子点标记生物分子的报道多为对大分子蛋白质的标记, 而对小分子肽标记的报道却很少.     

磺化修饰结合离子交换色谱和生物质谱富集鉴定含组氨酸肽段

通过在肽段的N端引入磺酸基,从而使含组氨酸的肽段与其他肽段在pH<3.0的条件下产生电荷差异,建立了一种基于强阳离子交换色谱(SCX)结合生物质谱富集鉴定含组氨酸肽段的方法,并以含有组氨酸的标准蛋白质为模型,进行了方法学考察。结果表明,经N端磺酸化后,含组氨酸的肽段能有效地被阳离子交换色谱富集,且在肽的N端引入磺酸基促进了肽的裂解,使之产生简单而信息丰富的二级质谱图,从而得到完美的质谱鉴定结果。这说明磺化修饰结合强阳离子交换色谱用于含组氨酸肽段的富集鉴定是可行的,且具有在蛋白质组研究中应用的潜力。     

High-performance liquid chromatography for the separation of angiotensin and its metabolites in human plasma and sweat

A reversed-phase high-performance liquid chromatography (HPLC) method with gradient elution for the separation of angiotensin peptides is described. The highly reproducible method allows the base-line separation of angiotensin peptides with UV detection at 225 nm. This chromatographic methodology in combination with radioimmunoassay (RIA) is used for the characterization of angiotensin peptides in human plasma and sweat.     

Sortase A-catalyzed peptide cyclization for the synthesis of macrocyclic peptides and glycopeptides

A chemoenzymatic method was developed for the synthesis of macrocyclic peptides and glycopeptides. Sortase A was found to mediate either head to tail cyclization or oligomerization and then head to tail cyclization of peptides and glycopeptides, depending on the peptide length, to produce 15-mer or higher cyclic peptides and glycopeptides.     

Prediction of protein secondary structure based on continuous wavelet transform

In this paper, continuous wavelet transform (CWT) is used to extract the number and the relevant positions of the α-helices and short peptides connecting α-helices and β-strands (connecting peptides) from the amino acid sequences of proteins. The amino acid sequence is first mapped into hydrophobicity sequence, and then transformed into CWT value of sequence domain in appropriate scale via CWT. The number and the relevant positions of the α-helices and connecting peptides can be extracted easily and accurately according to the minima of wavelet coefficient in corresponding CWT plot of hydrophobic value sequences with appropriate scale. The analytical results demonstrate that α-helices and connecting peptides can be predicted conveniently and rapidly when this method is used in the processing of 100 non-homologous sequences. However, this method is not suitable for predicting the length of α-helices and connecting peptides.     

A rapid label-free electrochemical detection and kinetic study of Alzheimer's amyloid beta aggregation

We present the first electrochemical detection, characterization, and kinetic study of the aggregation of Alzheimer's disease (AD) amyloid beta peptides (Abeta-40, Abeta-42) using three different voltammetric techniques at a glassy carbon electrode (GCE). This method is based on detecting changes in the oxidation signal of tyrosine (Tyr) residue. As the peptides aggregate, there are structure conformational changes, which affect the degree of exposure of Tyr to the molecular surface of the peptides. The results show significant differences in the aggregation process between the two peptides, and these correlate highly with established techniques. The method is rapid and label-free, and the principle can be universally applied to other protein aggregation studies related to diseases, such as Huntington's, Parkinson's, and Creutzfeldt Jacob (CJD). This method could also be explored in screening for the effectiveness of AD therapies.     

Amino acids and peptides. XXVII. Synthesis of phytochelatin-related peptides and examination of their heavy metal-binding properties

Phytochelatin (PC)-related peptides were prepared by a conventional solution method and their heavy metal-binding properties were examined. Different from the Cu2+ and Cu+ -binding properties of metallothionein (MT)-related peptides, the Cu2+ and Cu+ -binding properties of PC-related peptides were fairly dependent on structure. It is of interest that gamma-Glu-Cys-Gly (glutathione) exhibited quite different Cu2+ and Cu+ -binding properties from those of other PC-related peptides and its binding abilities were comparable to those of MT-related peptides. The Cd2+ -binding properties of glutathione were similar to those of Cys, and the Cd2+ -binding abilities of PC-related peptides increased in proportion to the increase of gamma-Glu-Cys peptide unit.