Current Organic Chemistry - Volume 7, Issue 15, 2003

Mass spectrometry has made outstanding progress in recent decades. By awarding the Nobel Prize for 2002 for chemistry to Drs. John B. Fenn and Koichi Tanaka, the scientific community has recognized the revolution mass spectrometry has made in identifying and analyzing large biological molecules. Therefore a dedicated issue on current topics in mass spectrometry is very appropriate. While writing this preface, I realized how global mass spectrometry has become. Seven articles included in this issue are written by seven research groups from seven different countries. The first article written by Kristina Hakansson, Helen J. Cooper, Robert R. Hudgins and Carol L. Nilsson presents an overall view of FTMS techniques. The second review by Thomas Brenna's group from Cornell University provides an excellent appraisal of high precision isotope ratio mass spectrometry. The third article takes mass spectrometry beyond earth. In this paper, John H. Bowie and his coworkers address the use of neutralization-reionization mass spectrometry in the study of gas phase anions. The next article on a related topic by Hans-Friedrich Grützmacher looks at radical cations. Rita Grandori' article addresses the utilization of electrospray ionization mass spectrometry in protein conformational studies. The sixth paper by Ohashi and Itoh discusses some unique ionization phenomena observed under FAB and MALDI conditions. The last article by Corinne Bure and Catherine Lange provides a comparison of CAD processes occurring in ESI sources and in traditional collision cells. I thank all authors for their combined effort. Articles gathered here are of interest to practicing mass spectrometrists as well to novices who would like to gain experience with mass spectrometry. I welcome any ideas and suggestions that will help me to improve the quality of future issues on this subject.

Tandem mass spectrometry (MS / MS) is a well-established technique for determining biomolecular primary sequences. The main advantages of MS / MS analysis compared to more traditional sequencing techniques are speed of analysis, sensitivity, high resolution and high mass accuracy. Currently, the highest performance (highest resolution, highest mass accuracy) mass analyzer is the Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The FT-ICR mass analyzer offers several alternative techniques for tandem mass spectrometry: for example “heating” techniques, such as sustained off-resonance irradiation collision-induced dissociation (SORI-CID), infrared multiphoton dissociation (IRMPD), blackbody infrared radiative dissociation (BIRD), and the recently introduced technique electron capture dissociation (ECD). In this review, we give an overview of FT-ICR theory, instrumentation, and performance. We also describe the different FT-ICR MS / MS techniques and discuss their capabilities and limitations for the structural biochemistry of peptides, proteins, oligonucleotides, carbohydrates, and glycoconjugates. For example, the complementarity of IRMPD and ECD for glycopeptide structural determination is demonstrated.

High precision isotope ratio mass spectrometry (IRMS) enables the detection of variability in isotope abundance due to natural processes. IRMS instruments are highly specialized for the analysis of 13 C / 12 C, 2 H / 1 H, 15 N / 14 N, 18 O / 16 O, and 34 S / 32 S via analysis gases CO 2, H 2, N 2, and SO 2 using a tight electron impact ion source, high transmission magnetic sector, and multiple collectors, delivering relative standard deviations of less than 0.01%. Recent developments exploiting continuous flow inlets have improved sample throughput and permitted a wide range of samples to be analyzed by highly automated systems. Isotope ratio analysis at the bulk mixture and compound-specific levels is now routine, and strategies for intramolecular analysis are on the horizon. Studies over 50 years have shown that isotopic fractionation due to physiological processes, specifically CO 2 transport processes within plants and photosynthesis, leads to variation in isotope ratio in natural compounds. Different chemical processes leading to isotope fractionation operate in the generation of petroleum. IRMS analysis can, in many cases, distinguish petroleum derived from different geographical regions that are otherwise identical. In wines and juices, climate differences that depend on geographic location induce isotopic fractionation that can be detected by IRMS and are characteristic of region and even vintage. Intramolecular or chiral isotope analysis is able to detect the origin of flavor compounds such as vanillin. Use of illicit, endogenous drugs such as testosterone, can be detected by isotope ratio analysis when referenced against another endogenous steroid upstream or independent of the endogenous biosynthetic pathway. These and related applications show that natural isotopic structure is a powerful tool for determining the origin of organic compounds.

In principle, Franck-Condon one-electron oxidation of an anion of known bond connectivity in a collision cell of a mass spectrometer may give the corresponding neutral. This neutral may be transmitted to a second collision cell in which it may be ionised. In order to be detected in the second cell, a neutral must be stable for the 10 -6 sec between formation in one collision cell and ionisation in the second. If the neutrals are converted to cations in the second cell, the positive ion fragmentations may, in principle, be used to probe the structure of the neutral(s) formed in the first collision cell, particularly if there is a peak corresponding to the ionised neutral. This technique is called neutralisation reionisation mass spectrometry, or specifically for the stepwise two-electron oxidation of an anion through a neutral to a cation, -NR+. The analogous -NRtechnique may also be used to study the neutral(s) formed in the first collision cell; in this case negative ion fragmentations are used to provide information concerning the structure(s) of the neutral(s). The -NR methods are techniques whereby transient and often reactive neutrals of unusual structures already detected (by rotational or infrared spectroscopy) in interstellar dust clouds or circumstellar clouds surrounding carbon-rich suns may be formed and investigated in the laboratory. Examples are described whereby -NR methods produce stable neutrals, e.g. the cumulenes C5H (this system is under further investigation), C7H2, and various oxo-cumulenes like CnO (n = 3, 5 and 7) and NCnO (n = 3 and 4). Some of these neutrals are known stellar molecules, others are awaiting experiments to test for their presence in either interstellar dust clouds or circumstellar envelopes. Some neutrals formed by -NR experiments have sufficient excess energy to undergo decomposition during the microsecond timeframe of the neutralisation reionisation experiment, e.g. energised oxo-cumulenes lose CO, and transient O2C-CO decomposes to CO and CO2. Other energised neutrals may rearrange to another isomer. The most interesting examples given in this article fall into this category. These include (i) the linear- C4 to rhombic-C4 rearrangement, and the analogous rearrangement of linear-C5 , both of which effectively randomise all carbons in these molecules, (ii) the complex atom scrambling of NCCCN, and (iii) those rearrangements where a neutral isomer rearranges to one which is more negative in energy, including the processes CCCHO to HCCCO, and C2COC2 to CCCCCO.

Radial cations of unsaturated organic hydrocarbons are reactive intermediates of many important chemical reactions. In particular, the rate constants of the reactions of these electron deficient species with electron rich reactants are increased by several orders of magnitude compared to the reaction between neutral partners. This effects has been termed “electron hole catalysis”. To get insight into the mechanism(s) of these reactions and the nature of the “electron hole catalysis” the reactions have been investigated in the gas phase using Fourier- transform ion cyclotron resonance (FT-ICR) mass spectrometry, and by molecular orbital calculations. The studies included the reactions of mono- and dihalogenated benzenes and naphthalenes as well as mono- and dihalogenated alkenes. Halogen substituents were chosen for practical reasons since the loss of a halogen substituent during the process can be conveniently used as a monitor reaction. The first part of this account discusses the methods used to determine the kinetics of ion / molecule reactions, in particular FT-ICR-mass spectrometry, and the special features of ion / molecule-reactions in the diluted gas phase. The second part deals with the reactions of halogenated arene radical cations with ammonia and amines as nucleophiles. The reactions correspond to a substitution of one halogen substituent by a radical cation mediated nucleophilic aromatic substitution and are only moderately efficient. Contrary to chemical intuition, the reaction efficiency decreases in the series Cl, Br, and I as substituent. Further, a peculiar dependance on the position of a second substituent is observed. Both effects can be explained by a mechanism involving at least the two steps of an addition of the nucleophile and of an elimination of the substituent and involving a reaction intermediate corresponding to a distonic onium ion. According to the Shaik / Pross model the first step is rate determining and depends strongly on the difference of the ionization energies of the reactants. A second effect comes from the dipole moment of the (neutral) dihalogenated arene, showing a decrease in the activation energy of the addition with an increase in dipole moment. The third part presents results of reactions of the radical cations of mono- and dihalogenated alkenes using ammonia, amines, and aliphatic alcohols as nucleophiles. These reactions are often efficient and may be collision controlled. Besides substitution of a halogen a variety of other reaction products is observed. The efficient reaction of alkene radical cations is in agreement with “electron hole catalysis”. An analysis by the Shaik / Pross model reveals that the initial addition step is now a very fast inner sphere electron transfer which occurs probably without any activation barrier. This is substantiated by molecular orbital calculations for selected reaction systems. Interestingly, experimental results show that addition of the nucleophile to the ionized C-C double bond competes even with intermolecular exothermic electron transfer and exothermic proton transfer. As a consequence, the ß-distonic onium ion, which is formed during the initial addition step, arises as a highly excited intermediate. The further course of the total reaction depends on the properties of this intermediate ß-distonic ion. The consequences of this reaction model for the individual reaction systems are discussed.

The possibility to study large molecules and their non-covalent interactions by mass spectrometry (MS) has opened novel ways to investigate protein folding and binding reactions. MS can be applied to protein conformational studies in two conceptually different ways. One approach uses MS to monitor mass changes produced by conformation-sensitive reactions, such as hydrogen / deuterium (H / D) exchange, alkylation and radiolysis. The second approach directly exploits the conformation dependence of the charge-state distributions (CSDs) of the multiply charged protein ions produced by electrospray-ionization (ESI). This review focuses on the information that has been provided by the latter kind of studies. An attempt is made to summarize and discuss the available evidence about the mechanism underlying this technique and its possible applications. The results of the studies described here include equilibrium and kinetic characterization of protein folding transitions and detection of folding intermediates. The case studies of myoglobin (Mb) and cytochrome c (cyt c) are discussed in particular detail. The unprecedented advantages offered by MS in the analysis of heterogeneous samples can now be applied to the study of dynamic systems involving different conformational states.

Flavin-derived compounds such as riboflavin, riboflavin 5'-phosphate (flavin mononucleotide, FMN) and flavin-adenine dinucleotide (FAD), under matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB) conditions produce unprecedented reduced species that can be attributed to [M+2H]+. and [M+3H]+ in addition to the anticipated [M+H]+ ions. On the other hand, electrospray ionization (ESI) conditions generate only the expected [M+H]+ as the sole molecular-related ion. A protonation from the matrix accompanied by a concomitant electron transfer, rather than the transfer of hydrogen atom(s), [H. of CH groups], is proposed to explain the observed reduction process. The reduction site expands to N(1)=C(10a)-C(4a)=N(5), a 1,4-diaza-1,3-butadiene system, in the isoalloxazine ring, resembling the behavior of prosthetic groups of aerobic dehydrogenases. Interestingly, CID-MS / MS of most abundant molecular-related ions generated by the FAB and ESI modes, namely [M+2H]+. for FAB and [M+H]+ for ESI, produce the identical even-electron fragment ion of isoalloxazine moiety, indicating a radical loss vs. the evenelectron molecule loss from the individual precursor ions. The observed phenomena are compared with those previously reported for FABMS, thermospray mass spectrometry (TSPMS) and ESIMS.

Electrospray ionization (ESI) is a soft ionization technique well suited for producing gas phase ions of biological macromolecules. The use of ESI when combined with collision-induced dissociation and mass analysis can give structural information about biomolecules. Traditionally, collisional fragmentation is carried out by tandem mass spectrometry. Several commercially available instruments permit tandem mass spectrometric experiments in space, in time or by post-source decay. Parameters influencing these processes and some applications are discussed in this article. It has been observed that in nearly all API sources, fragmentation of ions can arise by increasing the potential difference applied to the inlet and outlet orifices in the sampling region of the electrospray source kept under low vacuum. This process, called by variety of names, such as in-source collisioninduced dissociation (CID), up-front CID, cone-voltage CID or nozzle-skimmer dissociation, is less well understood than the traditional tandem mass spectrometric technique. Thanks to in-source CID, it is possible to obtain structural information with a single quadrupole instrument or to achieve a method close to MS / MS / MS with a triple quadrupole instrument. Results from a few studies comparing in-source CID in an electrospray ion source and CID in the collision gas cell of a tandem mass spectrometer have been published. These studies show that results from the both processes are generally comparable. The advantages and the drawbacks of each technique will be developed thereafter. In this article, the comparisons are reviewed and suggestions are made to help the reader to make a choice between the two fragmentation processes depending on the nature of the sample.