On-line electrogeneration of copper-peptide complexes in microspray mass spectrometry

The interaction of copper ions with peptides was investigated by electrospray mass spectrometry. Two electrospray micro-emitters were compared, the first one with a platinum electrode using a copper(II) electrolyte solution containing a peptide sample, and the second one with a sacrificial copper anode in a water/methanol solution containing only a peptide (i.e., angiotensin III, bradykinin, or Leu-enkephalin). The former yielded mainly Cu(2+) complexes either with histidine residues or with the peptide backbone (Cu(+) complexes can be also formed due to gas-phase reactions), whereas the latter can generate a mixture of both Cu(+) and Cu(2+) aqueous complexes that yield different complexation patterns. This study shows that electrospray emitters with soluble copper anodes enable the study of Cu(I)-peptide complexes in solution.     

Tandem mass spectrometry of amidated peptides

The behavior of C-terminal amidated and carboxylated peptides upon low-energy collision-induced dissociation (CID) was investigated. Two sets of 76 sequences of variable amino acid compositions and lengths were synthesized as model compounds. In most cases, C-terminal amidated peptides were found to produce, upon CID, an abundant loss of ammonia from the protonated molecules. To validate such MS/MS signatures, the studied peptides contained amino acids that can potentially release ammonia from their side chains, such as asparagine, glutamine, tryptophan, lysine and arginine. Arginine, and to a lesser extent lysine, was shown to induce a competitive fragmentation leading to the loss of ammonia from their side chains, thus interfering with the targeted backbone neutral release. However, when arginine or lysine was located at the C-terminal position mimicking a tryptic digest, losses of ammonia from the arginine side chain and from the peptide backbone were completely suppressed. Such results were discussed in the frame of peptidomic or proteomic studies in an attempt to reveal the presence of C-terminal amidated peptides or proteins.     

Studies of consecutive reactions of quinones in a reversed geometry mass spectrometer

Consecutive reactions involving the ejection of molecules of carbon monoxide from quinone systems have been studied. Using a reversed geometry double focusing mass spectrometer, the individual steps of a consecutive reaction may be separated into different field free regions. The structure of ions formed in the ion source may be compared with those formed from the fragmentation of metastable ions in a field free region by studying differences in the pattern of translational energies released when they break. In the case of anthraquinone it is found that two structures exist for the [M? CO]^+˙ ion. Critical energy measurements strengthen this argument. Furthermore, ¹⁸O labelling has been used to distinguish between the loss of carbon monoxide from different positions on the molecular ion of 1-hydroxyanthraquinone.     

Electrochemical processes in electrospray ionization mass spectrometry

Editorial Comment Last month we presented, as a Special Feature, a set of five articles that constituted a Commentary on the fundamentals and mechanism of electrospray ionization (ESI). These articles produced some lively discussion among the authors on the role of electrochemistry in ESI. Six authors participated in a detailed exchange of views on this topic, the final results of which constitute this month's Special Feature. We particularly hope that younger scientists will find value in this month's Special Feature, not only for the science that it teaches but also what it reveals about the processes by which scientific conclusions are drawn. To a degree, the contributions part the curtains on these processes and show science in action. We sincerely thank the contributors to this discussion. The give and take of intellectual debate is not always easy, and to a remarkable extent this set of authors has maintained good humor and friendships, even when disagreeing strongly on substance. Graham Cooks and Richard Caprioli Copyright 2000 John Wiley & Sons, Ltd.     

Design analysis of a laser ablation cell for inductively coupled plasma mass spectrometry by numerical simulation

A laser ablation setup including outer chamber, sample tube, sample holder and transport tubing was modelled and optimized using advanced computational fluid dynamics techniques. The different components of the setup were coupled and the whole device was modelled at once. The mass transport efficiency and transit times of near infrared femtosecond (fs) laser generated brass aerosols in pure argon and helium–argon mixtures were calculated at experimentally optimized conditions and a transient signal was constructed. The use of helium or argon did not influence the mass transport efficiency, but the signal structure changed. The signal fine structure was retrieved and experimentally validated. Bimodal peak structures were observed that seemed to originate from turbulent effects in the tubing connecting a Y-connector and the injector.     

Electrochemical simulation of redox reactions of glutathione

The effect of glutathione on the active oxygen reduction products at copper and platinum electrodes was studied by chronovoltammetry and pulse voltammetry. Electrochemical reduction of glutathione at the same electrodes was also examined. A mechanism was proposed for the reaction of glutathione with oxygen and its active forms, and attempts were made to simulate redox reactions initiating oxidative stress in vitro and glutathione effect thereon by electrochemical methods.     

有机化学反应中间体的质谱分析

有机化学反应中活性中间体的性质各异,对反应中间体的表征和研究对于阐明和确证反应机理、优化反应条件、筛选催化剂和发现新类型反应至关重要.近年来,质谱技术和在线微量采样技术的发展,极大程度地拓展了质谱学在该领域的应用.本文总结了近年来本领域发展的新策略、技术和方法,以及有机质谱技术在反应中间体研究中的应用.     

Charge permutation reactions in tandem mass spectrometry

Central to the tandem mass spectrometry experiment is the process that gives rise to product ions, i.e. the reaction intermediate to stages of mass analysis. Changes in mass or charge of the parent ion (or both) are generally readily detected by all forms of tandem mass spectrometry. Charge changing, or charge permutation, reactions have a long history in mass spectrometry. However, with the advent of new ionization methods, such as electrospray ionization, and the expansion of tandem mass spectrometry instrumentation to include ion trapping instruments, the past decade has seen a major increase in the types of charge permutation reactions that can be studied. Most charge permutation reactions involve electrons or protons as the charge mediating agents. This report, therefore, provides an overview of charge permutation reactions involving protons or electrons. Particular emphasis is placed on processes that involve interactions of precursor ions with gaseous neutral species, electrons or oppositely charged ions. Charge permutation reactions involving electron gain/loss are described first according to a rough order of the energy required for the reaction beginning with the most endoergic reactions and ending with the most exoergic reactions. An analogous approach is then taken with charge permutation reactions involving proton gain/loss. Important charge permutation reactions discussed herein, among others, include those referred to as charge inversion, charge stripping, electron capture dissociation, collision-induced ionization and charge separation. These reaction types, and others described herein, are the subjects of active research and are also finding use in many current areas of application. Copyright © 2004 John Wiley & Sons, Ltd.     

Studies of consecutive reactions of organic ions in a reversed geometry mass spectrometer

Over thirty consecutive reactions of a type m₁⁺ → m₂⁺ → m₃⁺ have been studied in a variety of organic compounds using a reversed geometry mass spectrometer. Two field free regions allow the separation of the two steps that make up the consecutive reaction sequence. Translational energy release measurements are used in the comparison of m₂⁺ ions formed as a result of a high energy process in the ion source with m₂⁺ ions formed as a product of a metastable decomposition. In some cases the structures of such ions have been found to be different. Examples have also been found where consecutive fragmentations of metastable ions do not occur although, when higher energy ions within the source are studied, the two-step reaction does take place. Furthermore, it has been found that a control over ion internal energy may be achieved by selecting portions of a peak due to the fragmentation of a metastable ion. Unimolecular reactions may then be used to study the reactivity of such ‘energy-selected’ ions; collision induced reactions can be used to study the structure of both the reactive and unreactive energy selected ions.     

Periodic operation of a tubular reactor: A simulation study of consecutive reactions in a chromatographic reactor

The performance of a chromatographic reactor is studied by means of computer simulations for consecutive reactions represented byA + B → RR + B → SFor square-wave pulsing it is demonstrated that a chromatographic reactor can give a higher yield than a plug-flow reactor operated at steady state if     

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.     

Accelerated reactions in field desorption mass spectrometry

Field desorption mass spectrometry under ambient conditions is used to study solution‐phase organic reactions in micro‐volumes. Reagent solution is transferred onto the microdendrites of the field emitter, and reaction products are examined online by mass spectrometry. Three reactions, hydrazone formation by phenyl hydrazine and indoline‐2,3‐dione, the Katritzky reaction between a pyrylium salt and anisidine, and the Hantzsch synthesis of 1,4‐dihydropyridine, were investigated, and reaction acceleration was observed to different extents. The increase in rate relative to the corresponding bulk reactions is attributed to solvent evaporation (simple concentration effect) and to the increase of surface‐to‐volume ratio (enhanced interfacial reactions). A distinguishing feature of this method of reaction acceleration, relative to that based on nano electrospray ionization, is the observation of radical cations and the formation of radical cation products. The study also breaks new ground in using field emitters at atmospheric pressure.     

Analysis of salivary peptides using HPLC-electrospray mass spectrometry

Salivary peptides are involved in a wide range of functions constituting the first line of defence of oral cavity and precursors of dental pellicle formation. The presence of mucins in saliva makes difficult the analysis of the proteic content. This is due mainly to aggregation phenomenon between mucins and other high molecular weight glycoproteins and salivary proteins. Considering the importance of salivary peptides in biological functions, we have evaluated the influence of four different extraction methodologies on the separation and identification of these proteins by HPLC-MS. Based on their molecular weight, we identified a total of 22 peptides when extraction was performed using a solution of guanidine (6 m), compared with 14 peptides identified when saliva is acidified with TFA, which is an often used procedure. Our results also show the presence of mucin bind peptides, which include statherin, PRP1, PRP3, Histatin 1 and Histatin 5.     

Electrospray ionization and tandem mass spectrometry of cysteinyl eicosanoids: Leukotriene C4 and FOG7

The cysteinyl leukotrienes, LTC4, LTD4 and LTE4, and the recently described cysteinyl eicosanoid, 5-oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG7) have been analyzed by tandem mass spectrometry. Both [M-H]- and [M+H]+ ions were produced by electrospray ionization and collision-induced dissociation of these molecular ion species were studied using both an ion trap and a triple quadrupole instrument. Product ion spectra obtained were characteristic of the structure of the cysteinyl leukotrienes and mechanisms of ion formation were investigated by using deuterium-labeled analogs. The product ion spectrum obtained following collision-induced dissociation of the [M-H]- anion from FOG7 was devoid of significant structural information and further studies of collision activation of the [M+H]+ spectrum were therefore examined. Positive ion MS3 spectra obtained in the ion trap from the gamma-glutamate cleavage products of FOG7 and its derivative (d7-FOG7) afforded an abundant ion not observed in spectra generated from the cysteinyl leukotrienes. Formation of this fragment ion likely occurred via a McLafferty-type rearrangement to afford cleavage of the C6-C7 bond adjacent to the sulfur atom and was valuable for the identification of the structure of FOG7 and defining the biosynthetic pathway as a 1,4-Michael addition of glutathione to 5-oxo-eicosatetraenoic acid (5-oxo-ETE).     

Computer-controlled sequencing of peptides by tandem mass spectrometry

A fully automated computer-controlled system was used to generate series of different linked scans at constant B2E and constant neutral loss in the second field-free region. This system has been shown to be suitable for deriving the amino acid sequence of oligopeptides.     

Chemical reactions in fast atom bombardment mass spectrometry

Examples of various chemical reactions occurring in the matrix or in the selvedge region in fast atom bombardment (FAB) mass spectrometry are discussed. These are categorized as oxidations and reductions; substitutions; clusterings and additions; and sample decomposition or transformation. Some reactions observed showed significant time behaviour and in one case it was possible to determine rate constants. These data suggest that chemical reactions can be accelerated significantly by fast atom bombardment.     

Chemical ionization mass spectrometry: Oligomerization reactions in tight ion sources

Association of carbonyl compounds with protons to give cluster ions can be observed in conventional chemical ionization mass spectrometry. The variation of the relative ion currents of proton bound ‘dimers’ formed with acetone and methylethyl ketone have been examined as a function of ion source partial pressures. Multiple ion/molecule reactions with N-methylol derivatives of amides and carbamates and repetitive losses of water give protonated oligomeric species. Ions having m/z values as large as 625 [M₆H? 5H₂O]⁺ have been detected in the chemical ionization spectrum of N-hydroxymethylurethane (M).     

Specific UV photodissociation of tyrosyl-containing peptides in multistage mass spectrometry

UV photodissociation (UVPD) at 262 nm has been carried out on protonated tyrosyl-containing peptides formed by trypsin digestion of apo-transferrin. Under UVPD, the main event is the fragmentation of the C(alpha)-C(beta) bond of the tyrosyl residues leading to a radical ion 107 Da below the precursor ion. The dissociation rate of this specific cleavage appears to be strongly dependent on the peptide sequence and is more prominent on the singly protonated species than on the doubly protonated state. The fragmentation spectra resulting from collisional activation of the protonated even-electron native peptides and of the odd-electron radical species prepared by UVPD are dominated by y-type backbone cleavages. A comparison of their respective y-ion pattern shows complementarities since the combination of both increases the sequence coverage of the peptide sequence. The specific detection of the neutral loss of 107 Da from peptides witnesses the content of at least one tyrosyl residue and, though preliminary, is proposed as a potential new filtering strategy during protein database searching.