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1.
Various classes of polypeptide antibiotics, including blocked linear peptides (gramicidin D), side-chain-cyclized peptides (bacitracin, viomycin, capreomycin), side-chain-cyclized depsipeptides (virginiamycin S), real cyclic peptides (tyrocidin, gramcidin S) and side-chain-cyclized lipopeptides (polymyxin B and E, amfomycin), were investigated by low-energy collision induced dissociation (LE-CID) as well as high-energy CID (HE-CID). Ion trap (IT) based instruments with different desorption/ionization techniques such as electrospray ionization (ESI), atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) and vacuum MALDI (vMALDI) as well as a vMALDI-time-of-flight (TOF)/curved field-reflectron instrument fitted with a gas collision cell were used. For optimum comparability of data from different IT instruments, the CID conditions were standardized and only singly charged precursor ions were considered. Additionally, HE-CID data obtained from the TOF-based instrument were acquired and compared with LE-CID data from ITs. Major differences between trap-based and TOF-based CID data are that the latter data set lacks abundant additional loss of small neutrals (e.g. ammonia, water) but contains product ions down to the immonium-ion-type region, thereby allowing the detection of even single amino-acid (even unusual amino acids) substitutions. For several polypeptide antibiotics, mass spectrometric as well as tandem mass spectrometric data are shown and discussed for the first time, and some yet undescribed minor components are also reported. De novo sequencing of unusually linked minor components of (e.g. cyclic) polypeptides is practically impossible without knowledge of the exact structure and fragmentation behavior of the major components. Finally, the described standardized CID condition constitutes a basic prerequisite for creating a searchable, annotated MS(n)-database of bioactive compounds. The applied desorption/ionization techniques showed no significant influence on the type of product ions (neglecting relative abundances of product ions formed) observed, and therefore the type of analyzer connected with the CID process mainly determines the type of fragment ions.  相似文献   

2.
    
The purpose of this work is to explore the mass fragment characterization of commonly used drugs through a novel approach, which involves isotope‐selective tandem mass spectrometry (MS/MS). Collision‐induced dissociation (CID) was performed with a low‐resolution linear ion trap mass spectrometer in positive electrospray ionization. Three pharmacologically active ingredients, i.e. omeprazole, meloxicam and brinzolamide, selected as model compounds in their own formulation, were investigated as a sodiated adduct [C17H19N3O3S + Na]+ (omeprazole) and as protonated adducts, [C14H13N3O4S2 + H]+ and [C12H21N3O5S3 + H]+, meloxicam and brinzolamide, respectively. Selecting a narrow window of ±0.5 m/z units, precursor ion fragmentation by CID‐MS/MS of isotopologues A + 0, A + 1 and A + 2 was found very useful to confirm the chemical formula of product ions, thus aiding the establishment of characteristic fragmentation pathways of all three examined compounds. The correctness of putative molecular formula of product ions was easily demonstrated by exploiting the isotope peak abundance ratios (i.e. IF+0/IF+1 and IF+0/IF+2) as simple constraints in low‐resolution MS instrumentations. Copyright © 2014 John Wiley & Sons, Ltd.  相似文献   

3.
A floating‐type low‐energy ion gun (FLIG) has been developed for high‐resolution depth profiling in ultrahigh vacuum (UHV). This UHV‐FLIG allows Ar+ ions of primary energy down to 50 eV to be provided with high current intensity. The developed UHV‐FLIG was sufficiently compact, being ～30 cm long, to be attached to a commercial surface analytical instrument. The performance of the UHV‐FLIG was measured by attaching it to a scanning Auger electron microprobe (JAMP‐10, Jeol), the base pressure of which in the analysis chamber was ～1 × 10?7 Pa. The vacuum condition of ～5 × 10?6 Pa was maintained during operation of the UHV‐FLIG without a differential pumping facility. Current density ranged from 41 to 138 µA cm?2 for Ar+ ions of primary energy 100–500 eV at the working distance of 50 mm. This ensures a sputtering rate of ～10 nm h?1 with 100 eV Ar+ ions for Si, leading to depth profiling of high resolution in practical use. Copyright © 2003 John Wiley & Sons, Ltd.  相似文献   

4.
    
The fragmentation of peptides and proteins upon collision‐induced dissociation (CID) is highly dependent on sequence and ion type (e.g. protonated, deprotonated, sodiated, odd electron, etc.). Some amino acids, for example aspartic acid and proline, have been found to enhance certain cleavages along the backbone. Here, we show that peptides and proteins containing dehydroalanine, a non‐proteinogenic amino acid with an unsaturated side‐chain, undergo enhanced cleavage of the N—Cα bond of the dehydroalanine residue to generate c‐ and z‐ions. Because these fragment ion types are not commonly observed upon activation of positively charged even‐electron species, they can be used to identify dehydroalanine residues and localize them within the peptide or protein chain. While dehydroalanine can be generated in solution, it can also be generated in the gas phase upon CID of various species. Oxidized S‐alkyl cysteine residues generate dehydroalanine upon activation via highly efficient loss of the alkyl sulfenic acid. Asymmetric cleavage of disulfide bonds upon collisional activation of systems with limited proton mobility also generates dehydroalanine. Furthermore, we show that gas‐phase ion/ion reactions can be used to facilitate the generation of dehydroalanine residues via, for example, oxidation of S‐alkyl cysteine residues and conversion of multiply‐protonated peptides to radical cations. In the latter case, loss of radical side‐chains to generate dehydroalanine from some amino acids gives rise to the possibility for residue‐specific backbone cleavage of polypeptide ions. Copyright © 2016 John Wiley & Sons, Ltd.  相似文献   

5.
    
Detection of nucleic acids and single nucleotide polymorphisms (SNPs) is of pivotal importance in biology and medicine. Given that the biological effect of SNPs often is enhanced in combination with other SNPs, multiplexed SNP detection is desirable. We show proof of concept of the multiplexed detection of SNPs based on the template‐directed native chemical ligation (NCL) of PNA‐probes carrying a metal tag allowing detection using ICP‐MS. For the detection of ssDNA oligonucleotides (30 bases), two probes, one carrying the metal tag and a second one carrying biotin for purification, are covalently ligated. The methodological limit of detection is of 29 pM with RSD of 6.7% at 50 pM (n = 5). Detection of SNPs is performed with the combination of two sets of reporter probes. The first probe set targets the SNP, and its yield is compared with a second set of probes targeting a neighboring sequence. The assay was used to simultaneously differentiate between alleles of three SNPs at 5‐nM concentration.  相似文献   

6.
    
In 1981 Spain went through a unique epidemic associated with a food-borne vector, affecting more than 20,000 people with over 800 deaths, which came to be known as the Toxic Oil Syndrome (TOS). Early epidemiological studies showed a link between this illness and the ingestion of rapeseed oil denatured with 2% aniline. This oil, originally aniline-denatured for industrial use, was fraudulently processed in an attempt to remove free aniline, and marketed as edible oil. Fatty acid anilides (FAA), monoesters and diesters of 3-(N-phenylamino)-1,2-propanediol (PAP) are present in oil samples as they arise in the refining process from reactions of aniline with constituent fatty acids and triglycerides of the oil matrix and are the only extraneous compounds found in these samples. To expand the search for the causative agents in TOS-associated oils and to look for new aniline-related compounds, an exhaustive characterization of laboratory-processed oils was undertaken. These oils, in the presence of aniline doped with 14C labelled aniline, were submitted to the laboratory conditions required for the generation of PAPs and FAAs. Laboratory-generated oil samples were submitted to a liquid-liquid extraction procedure to remove the unreacted aniline. The extract was processed by double solid-phase extraction to improve detection limits for minor amine-containing compounds in oils. The extracts enriched in aniline derivatives were submitted to on-line HPLC-UV-APCI-MS. Using two-dimensional ion maps, the components of several families of derivatives were readily identified. Additionally, the extracts were also fractionated by HPLC-UV and the fractions were analyzed by HPLC-APCI-MS/MS to obtain structural information. Standards of some of these compounds were synthesized and analyzed to confirm the results. A total of 115 aniline derivatives from 9 aniline-related families were identified in these oil samples. These included fatty acid anilides and an extensive array of phenylaminopropanediol esters distributed in eight major compound classes.  相似文献   

7.
    
Complex disulfide bond patterns in synaptosomal‐associated protein of 25 kD B (SNAP25B) are thought to regulate neurotransmitter release in response to oxidative stress. However, the steric feasibility of each possible disulfide pattern in SNAP25B has not been assessed. To assess the steric feasibility of hypothesized closely spaced complex disulfide patterning in SNAP25B and also the feasibility of identifying complex disulfide bond patterns with MS, we have developed a novel probabilistic analysis to unambiguously resolve complex double disulfide bond patterns by using an ion trap mass spectrometer. We analyzed fragmentation patterns of singly linked peptides to determine likely fragmentation events in an ion trap mass spectrometer and observed double and single backbone cleavage along with heterolytic cleavage of the disulfide bond. We modeled these same events in the doubly disulfide linked SNAP25B peptide and used a cumulative hypergeometric distribution with top–down scoring to both identify and differentiate these bonding patterns. Because of the presence of unique MS/MS peaks, two of the bonding patterns were directly identified. The third was assigned on the basis of full chromatographic separation and confirmed by modeling triple breakage fragments. In total, this work demonstrates the feasibility – and also limitations – of identification of complex intradisulfide patterns by using ion trap‐based collision‐induced dissociation‐based fragmentation methods. Copyright © 2013 John Wiley & Sons, Ltd.  相似文献   

8.
    
We have used tandem mass spectrometry (MS/MS)‐based analysis of glycopeptides in order to identify the composition and structure of rare glycoforms. The results illustrate utility of low‐energy MS/MS for structure identification. We have shown the presence of bifucosylated and trifucosylated glycoforms in human α‐1‐acid glycoprotein (AGP), a major plasma glycoprotein. Fucosylation in the case of AGP always occurs on the antennae; core fucosylation was not observed.  相似文献   

9.
The [M + 20H](20+)-[M + 12H](12+) charge states of native and reduced porcine elastase, a 25.9 kDa serine protease, were subjected to collisional activation in a quadrupole ion trap. For most charge states, ion parking was used to increase the number of parent ions over that yielded directly by electrospray. Ion-ion proton transfer reactions were used to reduce product ion charge states largely to +1 to simplify spectral interpretation. Both forms of the protein show charge state dependent fragmentation behavior. The native protein, which contains four disulfide linkages, shows almost no evidence for fragmentation within the regions of the protein linked by disulfide bonds. However, at the lowest charge states studied, evidence for cleavage of a least one of the disulfide bonds was evident in the appearance of a c-type ion. The highest charge states of native elastase showed several prominent cleavages C-terminal to valine residues. As the charge state decreased, however, preferential cleavages at acidic amino acid residues became important. The reduced form of the protein did not show particularly prominent cleavages at valine residues. However, many of the same preferential cleavages at acidic amino acid residues noted for the native protein were also observed in the same charge states of the reduced protein. The reduced protein also showed additional cleavages from regions of the protein that are ordinarily protected by disulfide linkages in the native form.  相似文献   

10.
    
The aim of this study was to investigate the fragmentation behavior induced by low‐energy collision‐induced dissociation (LE‐CID) of four selected antioxidants applied in lubricants, by two different types of ion trap mass spectrometers: a three‐dimensional ion trap (3D‐IT) and a linear IT (LIT) Orbitrap MS. Two sterically hindered phenols and two aromatic amines were selected as model compounds representing different antioxidant classes and were characterized by positive‐ion electrospray ionization (ESI) and LE‐CID. Various types of molecular ions (e.g. [M]+?, [M + H]+, [M + NH4]+ or [M + Na]+) were used as precursor ions generating a significant number of structurally relevant product ions. Furthermore, the phenolic compounds were analyzed by negative‐ion ESI. For both IT types applied for fragmentation, the antioxidants exhibited the same unusual LE‐CID behavior: (1) they formed stable radical product ions and (2) C? C bond cleavages of aliphatic substituents were observed and their respective cleavage sites depended on the precursor ion selected. This fragmentation provided information on the type of structural isomer usually not obtainable for branched aliphatic substituents utilizing LE‐CID. Comparing the two instruments, the main benefit of applying the LIT‐Orbitrap was direct access to elemental composition of product ions enabling unambiguous interpretation of fragmentation trees not obtainable by the 3D‐IT device (e.g. loss of isobaric neutrals). It should be emphasized that the types of product ions formed do not depend on the type of IT analyzer applied. For characterizing degradation products of antioxidants, the LIT‐Orbitrap hybrid system, allowing the determination of accurate m/z values for product ions, is the method of choice. Copyright © 2011 John Wiley & Sons, Ltd.  相似文献   

11.
    
A new tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source ‘ESI‐TOF/quadTOF’ was designed and constructed to achieve the desired aim of structural elucidation via high‐energy collision‐induced dissociation (CID), and the simultaneous detection of all fragment ions. The instrument consists of an orthogonal acceleration‐type ESI ion source, a linear TOF mass spectrometer, a collision cell, a quadratic‐field ion mirror and a microchannel plate detector. High‐energy CID spectra of doubly protonated angiotensin II and bradykinin were obtained. Several fragment ions such as a‐, d‐, v‐ and w‐type ions, characteristic of high‐energy CID, were clearly observed in these spectra. These high‐energy CID fragment ions enabled confirmation of the complete sequence, including leucine–isoleucine determinations. It was demonstrated that high‐energy CID of multiply protonated peptides could be achieved in the ESI‐TOF/quadTOF. Copyright © 2010 John Wiley & Sons, Ltd.  相似文献   

12.
    
The Claisen rearrangement is a well-known process occurring in condensed phase. In the gas-phase protonated allyl phenyl ethers, propargyl phenyl ethers, and N-allyl aniline produced by positive ion chemical ionization undergo Claisen rearrangement. This reaction has been observed even in the case of odd-electron molecular ions. Phenyl allenyl ether molecular ions actually undergo Claisen rearrangement, producing intense [M - CO](+*) ions. In this investigation, the behavior of protonated benzyloxy indole and some of its derivatives, obtained in electrospray conditions, is described. Low-energy MS/MS experiments carried out on [M + H](+) species show CO loss and an unexpected water loss: both can be justified only by the occurrence of Claisen rearrangement. Deuterium labeling experiments confirm this mechanism. The influence of different substituents in the indole moiety is discussed.  相似文献   

13.
建立了高效液相色谱-电喷雾离子阱质谱法(HPLC-ESI-ITMSn)同时测定了金银花中绿原酸和咖啡酸活性成分的分析方法。样品用体积分数80%乙醇回流提取,用Zorbax Eclipse XDB-C18柱分离,以体积分数0.5%甲酸的乙腈-甲酸水溶液为梯度流动相,以保留时间和质荷比对分离出的组分予以定性确证,用峰面积进行定量。绿原酸和咖啡酸的线性范围均为10.0~1000.0μg/L,检出限(以信噪比为3计)均为2.0μg/L。样品的加标平均回收率为92.7%~98.7%,相对标准偏差为1.4%~2.7%。该方法适用于其它复杂体系中绿原酸和咖啡酸的分析。  相似文献   

14.
    
In this work, the isolation step in the linear ion trap was performed using different “q values” conditions at a low collision-induced dissociation (CID) energy leading to the parent ion resolution improvements, reasonably due to better ion energy distribution. According to the results, we obtained a greater resolution and mass accuracy operating in both traditional electrospray and low voltage ionization near the q value = 0.778 and with a CID energy of 10%. This effect was evaluated with low-molecular-mass compounds (skatole and arginine). The proposed optimization yielded a superior instrument performance without adding technological complexity to mass spectrometry analyses.  相似文献   

15.
    
Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR/MS) offers the opportunity for gas phase cluster formation reactions at very low pressures and at temperatures that are different from room temperature. Reactions take place with single positive‐charge metal ions that are normally +2, +3, +4, etc., charged in solution. The ions formed are detected by measuring the current induced by their cyclotron rotation, but they cannot be physically separated and collected. Collision‐induced dissociation (CID) is widely used for ion‐structure determination via the fragmentation of the excited ions. CID study aims to determine the relationship between the Vpp [peak‐to‐peak voltage of the radiofrequency (rf) pulse] and the mass‐to‐charge (m/z) ratio, which will be used for the calculation of the center‐of‐mass translational kinetic energy (Ekcm) of the excited ion under investigation. CID studies are restricted to stable ions with relatively high abundance. Nevertheless, with the evolution of computational chemistry, such problems can be overcome whereby CID calculations will be used to provide the substantial parameters for computer software, such as the Gaussian 03 program, for the structure determination of the less stable NixS anions. The latter constitutes the core for our current research. © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2007  相似文献   

16.
    
Collision-induced dissociation (CID) of 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine was investigated using sequential tandem mass spectrometry. These adducts represent biomarkers of DNA damage linked to phenolic radicals and were investigated to gain insight into the effects of chemical structure of a C-8 modification on fragmentation pathways of modified 2'-deoxyguanosine (dG). CID in MS(2) of the deprotonated molecules of both the isomers generated the same product ion having the same m/z values. CID in MS(3) of the product ion at m/z 242 and CID in MS(4) experiments carried out on the selected product ions at m/z 225 and m/z 218 afford distinct fragmentation patterns. The conformational properties of isomeric product ions from CID showed that the ortho-isomers possess the unique ability to tautomerize through an intramolecular proton transfer between the phenolic OH group and the imine nitrogen (N7). Tautomerization of ortho-isomers to their keto-tautomers led to differences in their system of conjugated double bonds compared with either their enol-tautomer or the para-isomer. The charge redistribution through the N-7 site on the imidazole ring is a critical step in guanosine adduct fragmentation which is disrupted by the formation of the keto-tautomer. For this reason, different reaction pathways are observed for 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine. We present herein the dissociation and the gas-phase ion-molecule reactions for highly conjugated ions involved in the CID ion chemistry of the investigated adducts. These will be useful for those using tandem mass spectrometry for structural elucidation of C-8 modified dG adducts. This study demonstrates that the modification at the C-8 site of dG has the potential to significantly alter the reactivity of adducts. We also show the ability of tandem mass spectrometry to completely differentiate between the isomeric dG adducts investigated.  相似文献   

17.
We report here the generation of gas-phase complexes containing Pd(II), a ligand (deprotonated alanine, A-), and/or N-terminus derivatized peptides containing histidine as one of the amino acids. The species were produced by electrospray ionization, and their gas-phase reactions were investigated using ion-trap tandem mass spectrometry. Pd(II) forms a stable diaqua complex in the gas phase of the formula, [Pd(A-) (H(2)O)(2)]+, (where A- = deprotonated alanine) along with ternary complexes containing A- and peptide. The collision-induced dissociation (CID) patterns of the binary and ternary complexes were investigated, and the dissociation patterns for the ternary complexes suggest that: (a) the imidazole ring of the histidine side group may be the intrinsic binding site of the metal ion, and (b) the peptides fragment primarily by cleavage of the amide bond to the C-terminal side of the histidine residues. These observations are in accord with previous solution-state studies in which Pd(II) was shown to cause hydrolysis of an amide bond of a peptide at the same position.  相似文献   

18.
    
Electrospray‐generated precursor ions usually follow the ‘even‐electron rule’ and yield ‘closed shell’ fragment ions. We characterize an exception to the ‘even‐electron rule.’ In negative ion electrospray mass spectrometry (ES‐MS), 2‐(ethoxymethoxy)‐3‐hydroxyphenol (2‐hydroxyl protected pyrogallol) easily formed a deprotonated molecular ion (M‐H)? at m/z 183. Upon low‐energy collision induced decomposition (CID), the m/z 183 precursor yielded a radical ion at m/z 124 as the base peak. The radical anion at m/z 124 was still the major fragment at all tested collision energies between 0 and 50 eV (Elab). Supported by computational studies, the appearance of the radical anion at m/z 124 as the major product ion can be attributed to the combination of a low reverse activation barrier and resonance stabilization of the product ions. Furthermore, our data lead to the proposal of a novel alternative radical formation pathway in the protection group removal of pyrogallol. Copyright © 2009 John Wiley & Sons, Ltd.  相似文献   

19.
    
Diquaternary ammonium gemini surfactants have emerged as effective gene delivery vectors. A novel series of 11 peptide‐modified compounds was synthesized, showing promising results in delivering genetic materials. The purpose of this work is to elucidate the tandem mass spectrometric (MS/MS) dissociation behavior of these novel molecules establishing a generalized MS/MS fingerprint. Exact mass measurements were achieved using a hybrid quadrupole orthogonal time‐of‐flight mass spectrometer, and a multi‐stage MS/MS analysis was conducted using a triple quadrupole‐linear ion trap mass spectrometer. Both instruments were operated in the positive ionization mode and are equipped with electrospray ionization. Abundant triply charged [M+H]3+ species were observed in the single‐stage analysis of all the evaluated compounds with mass accuracies of less than 8 ppm in mass error. MS/MS analysis showed that the evaluated gemini surfactants exhibited peptide‐related dissociation characteristics because of the presence of amino acids within the compounds' spacer region. In particular, diagnostic product ions were originated from the neutral loss of ammonia from the amino acids' side chain resulting in the formation of pipecolic acid at the N‐terminus part of the gemini surfactants. In addition, a charge‐directed amide bond cleavage was initiated by the amino acids' side chain producing a protonated α‐amino‐ε‐caprolactam ion and its complimentary C‐terminus ion that contains quaternary amines. MS/MS and MS3 analysis revealed common fragmentation behavior among all tested compounds, resulting in the production of a universal MS/MS fragmentation pathway. Copyright © 2017 John Wiley & Sons, Ltd.  相似文献   

20.
    
A rapid, high resolution, and low sample consumption CZE method is developed for peptide nucleic acid (PNA) analysis for the first time. 30% v/v acetonitrile in PNA sample and 20% v/v acetonitrile in 50 mM borax‐boric acid (pH 8.7) as BGE were employed after optimization. The calibration curves were linear for PNA concentration ranging from 1 to 50 μmol/L. LOD and LOQ of PNA were 0.2 and 1.0 μmol/L, respectively. Since the commercially available reagent gives rise to huge PNA peak and an apparent impurity peak, the purity of PNA was evaluated to be about 81.4% by CZE method, obviously lower than the supplier's purity value of 99.9% evaluated by RP–HPLC, and also lower than 94.8% determined with RP–HPLC by our research group. The CZE method takes only 5 min, needs only 90 nL PNA, much less than 20 min and 20 μL PNA in RP–HPLC method. Moreover, the CZE method is applicable for the analysis of glutamic acid modified and lysine modified PNAs, they show different migration time with their corresponding complementary PNAs. Our results show CZE provides a new choice for PNA and modified PNA analysis, also their purity or quality evaluation.  相似文献   

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