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1.
Fast-atom bombardment mass spectrometry was used to study disulfide bonding patterns in heat-denatured human recombinant macrophage colony stimulating factor (rhM-CSF). The heat-denaturated protein was studied by analysis of the pattern of peptides in the proteolytic digests. Native rhM-CSF is a homodimer with intramolecular disulfide linkages between Cys7–Cys90, Cys48–Cys139, and Cys102–Cys146 and intermolecular linkages between Cys31-Cys31, and the pairs Cys157 and Cys159. Brief heating for 1 min leads to partial disulfide bond scrambling. In addition to the native disulfide bonds between Cys7–Cys90, Cys48–Cys139, and Cys31-Cys31, nonnative disulfide bonds were detected between Cys48–Cys90 and Cys48–Cys102. When heated for 5 min the disulfide bonds of rhM-CSF are completely scrambled and lead to nonnative intramolecular disulfide bonds between Cys48–Cys102 and Cys90–Cys102 and one intermolecular disulfide bond between Cys102–Cys102.  相似文献   

2.
Peptide and protein characterization by mass spectrometry (MS) relies on their dissociation in the gas phase into specific fragments whose mass values can be aligned as ‘mass ladders’ to provide sequence information and to localize possible posttranslational modifications. The most common dissociation method involves slow heating of even-electron (M+n H)n+ ions from electrospray ionization by energetic collisions with inert gas, and cleavage of amide backbone bonds. More recently, dissociation methods based on electron capture or transfer were found to provide far more extensive sequence coverage through unselective cleavage of backbone N–Cα bonds. As another important feature of electron capture dissociation (ECD) and electron transfer dissociation (ETD), their unique unimolecular radical ion chemistry generally preserves labile posttranslational modifications such as glycosylation and phosphorylation. Moreover, it was postulated that disulfide bond cleavage is preferred over backbone cleavage, and that capture of a single electron can break both a backbone and a disulfide bond, or even two disulfide bonds between two peptide chains. However, the proposal of preferential disulfide bond cleavage in ECD or ETD has recently been debated. The experimental data presented here reveal that the mechanism of protein disulfide bond cleavage is much more intricate than previously anticipated.  相似文献   

3.
Recently we have shown that, as a versatile ionization technique, desorption electrospray ionization (DESI) can serve as a useful interface to combine electrochemistry (EC) with mass spectrometry (MS). In this study, the EC/DESI-MS method has been further applied to investigate some aqueous phase redox reactions of biological significance, including the reduction of peptide disulfide bonds and nitroaromatics as well as the oxidation of phenothiazines. It was found that knotted/enclosed disulfide bonds in the peptides apamin and endothelin could be electrochemically cleaved. Subsequent tandem MS analysis of the resulting reduced peptide ions using collision-induced dissociation (CID) and electron-capture dissociation (ECD) gave rise to extensive fragment ions, providing a fast protocol for sequencing peptides with complicated disulfide bond linkages. Flunitrazepam and clonazepam, a class of nitroaromatic drugs, are known to undergo reduction into amines which was proposed to involve nitroso and N-hydroxyl intermediates. Now in this study, these corresponding intermediate ions were successfully intercepted and their structures were confirmed by CID. This provides mass spectrometric evidence for the mechanism of the nitro to amine conversion process during nitroreduction, an important redox reaction involved in carcinogenesis. In addition, the well-known oxidation reaction of chlorpromazine was also examined. The putative transient one-electron transfer product, the chlorpromazine radical cation (m/z 318), was captured by MS, for the first time, and its structure was also verified by CID. In addition to these observations, some features of the DESI-interfaced electrochemical mass spectrometry were discussed, such as simple instrumentation and the lack of background signal. These results further demonstrate the feasibility of EC/DESI-MS for the study of the biology-relevant redox chemistry and would find applications in proteomics and drug development research.  相似文献   

4.
Manabe T  Jin Y 《Electrophoresis》2005,26(1):257-267
In the course of searching methods to extract proteins from Coomassie blue-stained polyacrylamide gels, we found proteins are extracted in relatively high recovery when the gel pieces are soaked in alkaline solutions. However, alkaline conditions are known to cause decomposition of proteins, especially peptide bond cleavage and disulfide degradation. We studied the effects of alkaline on two purified proteins, chicken insulin and bovine alpha-lactalbumin, both containing four disulfide bonds in their structure. The process of covalent bond cleavage was traced by analyzing the mass spectra of the proteins using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). When the proteins are kept at pH 13 in the presence of 0.1% dithithreitol (DTT), peptide bonds at the C-terminal side of asparaginyl residues are preferably cleaved producing succinimides, whereas cysteinyl residues are not decomposed. In the absence of DTT, the disulfide bonds of the proteins are decomposed by alkaline and the cleavage of the peptide bonds are less obvious, possibly because the conformation of the proteins are partially retained until the full decomposition of disulfide bonds. These results identified for the first time the cleavage sites of proteins under alkaline treatment and further suggested the general tendency of the reactions, both in the presence and absence of DTT.  相似文献   

5.
蛋白质中二硫键的定位及其质谱分析   总被引:2,自引:0,他引:2  
二硫键是一种常见的蛋白质翻译后修饰,对稳定蛋白质的空间结构,保持及调节其生物活性等都有着非常重要的作用。因此,确定二硫键在蛋白质中的位置是全面了解含二硫键蛋白化学结构的重要方面。在众多实验方法中,现代质谱技术因其操作简单、快速、灵敏等优点而成为分析二硫键的重要手段。本文介绍了目前主要的定位二硫键的方法,以及质谱在二硫键定位分析中的应用与进展。  相似文献   

6.
Desfuroylceftiofur (DFC) is a bioactive beta-lactam antibiotic metabolite that has a free thiol group. Previous experiments have shown release of DFC from plasma extracts after addition of a disulfide reducing agent, suggesting that DFC may be bound to plasma and tissue proteins through disulfide bonds. We have reacted DFC with [Arg(8)]-vasopressin (which has one disulfide bond) and bovine insulin (which has three disulfide bonds) and analyzed the reaction products by use of electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS), which has previously shown preferential cleavage of disulfide bonds. We observe cleavage of DFC from vasopressin and insulin during ECD, suggesting that DFC is indeed bound to peptides and proteins through disulfide bonds. Specifically, we observed dissociative loss of one, as well as two, DFC species during ECD of [vasopressin + 2(DFC-H) + 2H](2+) from a single electron capture event. Loss of two DFCs could arise from either consecutive or simultaneous loss, but in any case implies a gas phase disulfide exchange step. ECD of [insulin + DFC + 4H](4+) shows preferential dissociative loss of DFC. Combined with HPLC, ECD FT-ICR-MS may be an efficient screening method for detection of drug-biomolecule binding.  相似文献   

7.
We have used on-line sample clean-up, concentration, and chromatography with electrospray ionization mass spectrometry (ESI-MS), to characterize and determine the presence of disulfide bonds in recombinant full-length rat brain calbindin D28K and two deletion mutants of the protein, one lacking EF-hand 2 (calbindin delta 2) and the other lacking EF-hands 2 and 6 (calbindin delta 2,6). The molecular weights of the expressed proteins dissolved in biological buffers were determined with high accuracy using a low-flow, pressurized chamber infusion system, that allows on-line protein clean-up by removing buffers/salts incompatible with ESI-MS. The molecular weight determinations showed that the amino-terminal methionine residues had been cleaved during the expression and isolation of the recombinant proteins. Approximately 85-90% of the protein sequences were confirmed by on-line HPLC-ESI-MS analysis of peptides generated by a lysyl endoproteinase C digestion. Comparisons of ESI-MS spectra of native and reduced calbindin D28K and delta 2 show that the full length- and delta 2 mutant-protein contain one disulfide bond. Molecular mass determinations of calbindin delta 2,6 showed that this protein contains a highly active cysteine residue that covalently binds a mercaptoethanol group, or forms a homodimer via a disulfide bond. The results show surprising differences amongst the deletion mutants of calbindin D28K with respect to the formation of disulfide bonds. These differences are not readily detected by other techniques and show that ESI-MS is a powerful, rapid method by which to detect disulfide linkages for intact proteins.  相似文献   

8.
A peptide containing a single disulfide bond was sequenced using high-energy collision-induced dissociation (HE-CID) in conjunction with a high mass resolution time-of-flight tandem mass spectrometer equipped with a matrix-assisted laser desorption/ionization source. This mass spectrometer, which has spiral ion trajectory, allowed both high mass resolution and high precursor ion selectivity. It is difficult to obtain sufficient product ions from peptides containing disulfide bonds using HE-CID due to the single collision in the gas phase. To compensate for insufficient dissociation, the disulfide bond was cleaved via an in-source reduction process using 1,5-diaminonaphthalene, a reducing matrix. After applying the reduction in the ionization, subsequent sequencing using HE-CID provided the detailed structural information of the peptide containing the single disulfide bond.  相似文献   

9.
The study of noncovalent interactions by mass spectrometry has become an active field of research in recent years. The role of the different noncovalent intermolecular forces is not yet fully understood since they tend to be modulated upon transfer into the gas phase. The hydrophobic effect, which plays a major role in protein folding, adhesion of lipid bilayers, etc., is absent in the gas phase. Here, noncovalent complexes with different types of interaction forces were investigated by mass spectrometry and compared with the complex present in solution. Creatine kinase (CK), glutathione S-transferase (GST), ribonuclease S (RNase S), and leucine zipper (LZ), which have dissociation constants in the nM range, were studied by native nanoelectrospray mass spectrometry (nanoESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking (XL). Complexes interacting with hydrogen bonds survived the transfer into gas phase intact and were observed by nanoESI-MS. Complexes that are bound largely by the hydrophobic effect in solution were not detected or only at very low intensity. Complexes with mixed polar and hydrophobic interactions were detected by nanoESI-MS, most likely due to the contribution from polar interactions. All noncovalent complexes could easily be studied by XL MALDI-MS, which demonstrates that the noncovalently bound complexes are conserved, and a real “snap-shot” of the situation in solution can be obtained.  相似文献   

10.
The disulfide bonding patterns in the N-terminal (LN) domains of the basement membrane protein laminin beta1 have not been investigated so far. We report an in-depth mass spectrometric analysis using offline nano-high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (nano-HPLC/MALDI-TOF/TOF-MS) for determining the disulfide bond patterns in the LN-domain of recombinant mouse laminin beta1 chain for the first time. Mass spectra were recorded and the putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the disulfide bond. Screening the fragment ion mass spectra of disulfide-linked peptides for characteristic 66-amu patterns (34 u +32 u), arising from symmetric and asymmetric cleavage of disulfide bonds, facilitated their identification. Using various enzymes for proteolytic digestion of a recombinant laminin beta1 chain N-terminal protein fragment, a linear bonding pattern of the eight cysteine residues in the LN-domain of the laminin beta1 chain was observed with a (1-2, 3-4, 5-6, 7-8) connectivity of cysteines. The identical disulfide-bonding pattern was found in E4, the N-terminal laminin beta1 chain fragment derived by elastase digestion of mouse tumor laminin-111, confirming that this pattern also occurs in native laminin.  相似文献   

11.
二硫键是一种与多肽及蛋白质结构和功能密切相关的化学键. 当多肽中存在多个半胱氨酸时, 形成的二硫键可能会存在多种配对方式. 快速且精准地定位多肽中多对二硫键对研究多肽的结构与功能间的关系十分重要. 本文开发了一种基于化学裂解和生物质谱的新方法, 对利那洛肽中3对二硫键进行了精准定位. 通过解析裂解后特异肽段的二级质谱图, 确定利那洛肽中3对二硫键的配对方式分别为Cys1-Cys6, Cys2-Cys10和Cys5-Cys13. 该方法为二硫键的定位研究提供了新思路.  相似文献   

12.
The peptide library present in the venom of the piscivorous marine snail Conus achatinus has been probed using a combination of mass spectrometry and cDNA sequencing methods. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis, before and following global reduction/alkylation of peptide mixtures, permits the rapid classification of individual components on the basis of the number of disulfide bonds. Mass fingerprinting and the reverse phase HPLC retention times permit a further deconvolution of the library in terms of peptide size and hydrophobicity. Sequencing of cDNA derived using O-superfamily specific primers yielded five complete conotoxin precursor sequences, ranging in polypeptide length from 75-87 residues containing six Cys residues at the C-terminus. Sequence analysis permits classification of the five putative mature peptides (Ac 6.1 to Ac 6.5) as delta, omega, and omega-like conotoxins. The presence of these predicted peptides in crude venom was established by direct matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) sequencing following trypsin digestion of the peptide mixture after global reduction/alkylation. The determination of partial peptide sequences and comparison with the predicted sequences resulted in the identification of four of the five predicted conotoxins. The characterization of posttranslationally modified analogs, which are hydroxylated at proline or amidated at the C-terminus is also demonstrated. Crude venom analysis should prove powerful in studying both inter- and intra-species variation in peptide libraries.  相似文献   

13.
In our laboratory, we have been studying the reductive processes that occur during matrix-assisted laser desorption/ionization (MALDI) experiments. Recently, we have finished an analysis of the DHB matrix effect on the azo group in cyclic peptides. However, deep understanding of disulfide bond behaviour during a mass spectrometry (MS) experiment is much more important in proteomics as its reduction can cause serious errors in protein spectra interpretation. Therefore, we have focused on intra- and intermolecular disulfide bonds as well as disulfide bonds connecting cysteine and 2-thio-5-nitrobenzoic acid (TNB, Ellman's reagent modification) in model peptides during MALDI MS measurements. While the reduction was not observed for intra- and intermolecular cysteine-cysteine disulfide bonds, the disulfide connection between cysteine and TNB was always affected. It was proved that TNB and Ellman's reagent can act as a matrix itself. The results obtained enabled us to propose a reaction mechanism model which is able to describe the phenomena observed during the desorption/ionization process of disulfide-containing molecules.  相似文献   

14.
The disulfide bond pattern of Trimeresurus stejnegeri lectin (TSL), a new member of the C-type lectin family, was determined by mass spectrometry. Four intrachain disulfide bonds of TSL, Cys(3)-Cys(14), Cys(31)-Cys(131), Cys(38)-Cys(133) and Cys(106)-Cys(123), and two interchain linkages, Cys(2)-Cys(2) and Cys(86)-Cys(86), were determined. Three strategies were used in this work. One intrachain (Cys(106)-Cys(123)) and one interchain (Cys(86)-Cys(86)) disulfide linkages were detected by standard MS methods. The disulfide bonds Cys(2)-Cys(2) and Cys(3)-Cys(14) were analyzed using a modified partial reduction procedure and MS/MS. The last two disulfide bonds were characterized by a MS/MS/MS technique. The strategies developed in this work could be applied more generally to detection of disulfide bond patterns.  相似文献   

15.
The gas‐phase free radical initiated peptide sequencing (FRIPS) fragmentation behavior of o‐TEMPO‐Bz‐conjugated peptides with an intra‐ and intermolecular disulfide bond was investigated using MSn tandem mass spectrometry experiments. Investigated peptides included four peptides with an intramolecular cyclic disulfide bond, Bactenecin (RLC RIVVIRVC R), TGF‐α (C HSGYVGVRC ), MCH (DFDMLRC MLGRVFRPC WQY) and Adrenomedullin (16–31) (C RFGTC TVQKLAHQIY), and two peptides with an intermolecular disulfide bond. Collisional activation of the benzyl radical conjugated peptide cation, which was generated through the release of a TEMPO radical from o‐TEMPO‐Bz‐conjugated peptides upon initial collisional activation, produced a large number of peptide backbone fragments in which the S? S or C? S bond was readily cleaved. The observed peptide backbone fragments included a‐, c‐, x‐ or z‐types, which indicates that the radical‐driven peptide fragmentation mechanism plays an important role in TEMPO‐FRIPS mass spectrometry. FRIPS application of the linearly linked disulfide peptides further showed that the S? S or C? S bond was selectively and preferentially cleaved, followed by peptide backbone dissociations. In the FRIPS mass spectra, the loss of ?SH or ?SSH was also abundantly found. On the basis of these findings, FRIPS fragmentation pathways for peptides with a disulfide bond are proposed. For the cleavage of the S? S bond, the abstraction of a hydrogen atom at Cβ by the benzyl radical is proposed to be the initial radical abstraction/transfer reaction. On the other hand, H‐abstraction at Cα is suggested to lead to C? S bond cleavage, which yields [ion ± S] fragments or the loss of ?SH or ?SSH. Copyright © 2011 John Wiley & Sons, Ltd.  相似文献   

16.
采用凝胶色谱、高效液相色谱等方法从栖息于我国南海的桶形芋螺的毒液中纯化出一个新的芋螺毒素BtIIIB.采用氨基酸组成分析、质谱分析和Edman降解测定BtIIIB为15肽, 其氨基酸序列为: CCELPCHGCVPCCWP.结构中含有6个半胱氨酸, 形成3对分子内二硫键.采用部分还原的方法, 分步还原毒素中的二硫键, 用氰基化试剂衍生生成巯基, 然后在碱性条件下将衍生的产物进行裂解, 用MALDI-TOF MS测定裂解后片段的分子量, 确定了其二硫键的配对方式为Cys1-Cys13, Cys2-Cys9, Cys6-Cys12的部分交叉式结构, 是芋螺毒素中比较特别的配对方式.  相似文献   

17.
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.  相似文献   

18.
A novel electrochemical (EC) method for fast and efficient reduction of the disulfide bonds in proteins and peptides is presented. The method does not use any chemical agents and is purely instrumental. To demonstrate the performance of the EC reactor cell online with electrospray mass spectrometry, insulin and somatostatin were used as model compounds. Efficient reduction is achieved in continuous infusion mode using an EC reactor cell with a titanium-based working electrode. Under optimized conditions, the presented method shows almost complete reduction of insulin and somatostatin. The method does not require any special sample preparation, and the EC reactor cell makes it suitable for automation. Online EC reduction followed by collision-induced dissociation fragmentation of somatostatin showed more backbone cleavages and improved sequence coverage. By adjusting the settings, the EC reaction efficiency was gradually changed from partial to full disulfide bonds reduction in α-lactalbumin, and the expected shift in charge state distribution has been demonstrated. The reduction can be controlled by adjusting the square-wave pulse, flow rate or mobile phase composition. We have shown the successful use of an EC reactor cell for fast and efficient reduction of disulfide bonds for online mass spectrometry of proteins and peptides. The possibility of online and gradual disulfide bond reduction adds a unique dimension to characterization of disulfide bonds in mid- and top-down proteomics applications.
Figure
Principle of electrochemical reduction of disulfide bonds in proteins  相似文献   

19.
The ability of a thiol‐containing molecule, thiosalicylic acid (TSA), to function as a reactive matrix for matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry analysis of peptides has been investigated. Although TSA has reducing characteristics, the use of TSA did not cause a reduction‐induced MALDI in‐source decay, probably because of the weak interactions between the thiol group in TSA and the carboxyl oxygen in the peptide. In contrast, when peptides containing disulfide bonds were analyzed by MALDI with TSA as the matrix, the disulfide bond was partially cleaved owing to the reaction with TSA, producing TSA‐adducted peptides. The reaction between the disulfide bond and TSA was suggested to be occurred in solution. The comparison of the MALDI mass spectra obtained using conventional matrix and TSA allows us to count the number of disulfide bonds in the peptides. Copyright © 2017 John Wiley & Sons, Ltd.  相似文献   

20.
Potentially biologically-active nanostructures can be created from single chains of unmodified peptides by cross-linking different regions of the chain by disulfide bonds and cleaving the chain at specified sites to obtain the final configuration. The availability of techniques for assembly and characterization of such structures was tested on a two-loop structure created from a 21-residue linear peptide. Directed intra-molecular disulfide bond formation was performed by inserting partial sequences favoring intra-molecular SS bond formation ("loops") separated by partial sequences disfavoring such a process ("spacers") into the precursor sequence. Peptide bond cleavage by partial acid hydrolysis at specific sites (GG, NP/DP) inside the loops opened them; the same process in the spacer separated the loops. Synthesis, oxidation and bond cleavage were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). The hydrolysis fragments of the produced nanostructures were characterized by tandem electrospray ionization Fourier transform mass spectrometry (ESI FT-MS) with collisional and electron capture dissociations. The latter technique was especially useful as it cleaves SS bonds preferentially. The feasibility of the proposed synthesis approach and the adequacy of the analysis techniques for the test structure were demonstrated.  相似文献   

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