Tandem mass spectrometry structural information obtained from

In the last decade mass spectrometry (MS) has been revolutionized by new methods for ionizing large molecules, even as large as trypsin, molecular weight (MW) 23,463 (1). Increasing the size of an unknown molecule results in an exponential increase in the structural information needed for its identification (2-4). MS is already well recognized for the unusual amount of information obtainable from picomole samples, and high resolution and tandem MS (MS/MS) (5) can provide substantially more information. 

Tandem mass spectrometry allows more structural information to be obtained on a particular ionic species, because the used ionization method yields relatively few structurally diagnostic fragments, or because its fragmentation is obscured by the presence of other compounds in the mixture introduced in the source, or because it is obscured by other ions generated from the matrix in the course of ionization. 

In practical application scanning can be manipulated on-the-fly within a chromatographic separation to obtain maximum information. In metabolism studies or as a chemical dosimeter, the structural feature of the parent compound and its unique neutral loss occurring on collisional activation, marks the metabolite. The mass of the metabolite is then obtained from the TSP mass spectrum at Q1 and the product ion spectrum of the metabolite molecule ion is obtained by product ion scanning. Recent publications have discussed additional applications of both tandem mass spectrometry (26. 271 and thermospray tandem mass spectrometry (28) in metabolite structure elucidation. 

Both ESI and APCI generate molecular ions from polar and labile biomaterials with remarkable ease and efficiency. But the amount of structural information that can be deduced from ESI spectra, in particular, is rather limited. Thus, only the use of tandem mass spectrometry (MS/MS) together with liquid chromatography opens further dimensions in the field of bio-organic analysis (Fig. 1). Beside retention data and UV spectra one can obtain both molecular mass and substructure specific information of any analyte out of a complex matrix. 

In addition, new tandem mass spectrometry technologies were also among the important innovations. Apart from traditional collision-induced dissociation (CID) [89-91], a variety of activation methods (used to add energy to mass-selected ions) based on inelastic collisions and photon absorption have been widely utilized. They include IR multiphoton excitation [92,93], UV laser excitation [94—97], surface-induced dissociation (SID) [98-100], black body radiation (101, 102], thermal dissociation [103], and others. As the fragmentation of peptide/protein ions is a central topic in proteomics, there is strong interest in such novel ion dissociation methods as electron capture dissociation (ECD) [104, 105] and electron transfer dissociation [22]. These new methods can provide structural information that complements that obtained by traditional collisional activation. Also, very recently, ambient ion dissociation methods such as atmospheric pressure thermal dissociation [106] and low temperature plasma assisted ion dissociation [107] have been reported. 

The feasibility of on-line electrochemistry mass spectrometry in the study of electrode processes has recently been demonstrated by Heitbaum et al. . We have tested the potential of on-line mass spectrometry in the study of redox reactivity of biological compounds with uric acid as a probe. Electrochemical oxidation of uric acid has been studied extensively . The scheme in Figure 6 shows the electrochemical oxidation pathway of uric acid and indicates intermediates and products which were identified by on-line electrochemistry thermospray mass spectrometry (EC/TSP/MS/MS) . In our studies, tandem mass spectrometry (MS/MS) was used to obtain structurally informative fragmentation patterns (daughter spectra) of standards for comparison to the mass spectra of intermediates and products obtained by EC/TSP/MS/MS. This, for example, allowed identification of allantoin through its characteristic daughter spectrum. It also allowed confirmation of the structural features of the intermediate, bicyclic carboxylic acid, which apparently forms from the imine alcohol in the oxidation of uric acid. The intermediates and products which were identified in this way are indicated in the scheme, and mass spectral results are summarized in Table 1. 

In addition to an efficient scan operation mode, ion trap mass spectrometers offer possibilities of MS" experiments. With the selective removal of ions from the trap, storage of a precursor ion of interest and its resonant activation,collision-induced dissociation (CID) is obtained, giving rise to product ion spectra that provide information comparable to triple quadrupole MS/MS experiments. Moreover, product ions obtained from CID can be further subjected to isolation and dissociation, which enables MS" analyses and, thus, more insights into structural features of analytes and gas-phase reaction and degradation processes. However, also limitations of ion trap tandem mass spectrometry were reported primarily resulting from the low-mass cut-off in MS" experiments, which were circumvented for instance by combining in-space dissociation devices with ion trap mass analyzers. 

As an instrumental approach to conventional electrophoresis, capillary electrophoresis offers the capability of on-line detection, micropreparative operation and automation (6,8,45-47). In addition, the in tandem connection of capillary electrophoresis to other spectroscopy techniques, such as mass spectrometry, provides high information content on many components of the simple or complex peptide under study. For example, it has been possible to separate and characterize various dynorphins by capillary electrophoresis-mass spectrometry (33). Therefore, the combination of CE-mass spectrometry (CE-MS) provides a valuable analytical tool useful for the fast identification and structural characterization of peptides. Recently, it has been demonstrated that the use of atmospheric pressure ionization using Ion Spray Liquid Chromatography/ Mass Spectrometry is well suited for CE/MS (48). This approach to CE/MS provides a very effective and straightforward method which allow the feasibility of obtaining CE/MS data for peptides from actual biological extracts, i.e., analysis of neuropeptides from equine cerebral spinal fluid (33). 

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