With the development of soft ionization techniques and the advances in instruments, mass spectrometry has come to be an essential tool for the characterization of proteins. It has been used successfully for the investigation of the amino acid sequences of large macromolecules, such as IgG. In addition, it is effective for the characterization of post-translational modifications of proteins.
The tertiary structure of proteins has generally been investigated using X-ray crystallography and NMR. Precise information on the tertiary structure can be obtained by these techniques. However, these analyses are time consuming and require a large scale sample preparation. Accordingly, it is impossible to apply these methods directly to a small amount of proteins derived from the living organisms. On the other hand, mass spectrometry requires only a small amount of sample, such as several pico-mole, for the determination of the molecular weight of a protein and/or a peptide in a routine analysis. Consequently, studies on the tertiary structure and/or the surface structure of the protein molecules have come to be carried out by mass spectrometry, in order to characterize the tertiary and/or surface structure of the protein with a small amount of sample.
This paper discusses the characterization not only of the primary structure but also of the tertiary structure of proteins by mass spectrometry.

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The effect on the fragmentations of N-benzyloxycarbonyl-protected tri and tetrapeptide ethyl esters due to the existence of L-proline was examined in field desorption, fast atom bombardment and tandem mass spectrometry. A significant difference was found among the abundances of the molecular-related ion and the fragment ions formed by the cleavage of the benzyloxycarbonyl group, depending on the numbers and positions of prolyl residues in the derivatives. The results showed that the conformational difference in peptide molecules due to the existence of Pro and the existence of the amide protons near the benzyloxycarbonyl group in the peptides have an effect on the fragmentations of molecules in field desorption and fast atom bombardment mass spectrometry.

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Fragmentation of N₂O⁺ in the narrow energy regime of 14.8 to 15.5 eV, where electronic predissociation occurs involving a state transition between N₂O⁺ (X₂II_i) and N₂O⁺ (2⁴Σ^-), has been studied by threshold photoelectron-photoion coincidence (TPEPICO) mass spectrometry using a non-pulsed ion-extraction field. Detailed breakdown curves and kinetic energy releases accompanied by fragmentation were obtained. Kinetic energy releases observed for both the NO⁺ and O⁺ formations were consistent with the predictions based on the spectator model of direct dissociation. Results obtained have thus suggested that the fragmentations on the N₂O⁺ (2⁴Σ^-) state occur considerably faster than the rate with which the system jumps from X²II_i to 2⁴Σ^-, and therefore this transition probability has been given approximately by the summation of both the NO⁺ and O⁺ breakdown curves with a most probable energy of ca. 15.2 eV in the curve-crossing.

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In order to understand differences in fragmentation between [M+H]⁺ and [M+A]⁺ ions (A=Li, Na, K, Rb, and Cs) in more detail, dissociation reactions of [M+H]⁺ and [M+Li]⁺ ions of N-t-butoxycarbonyl (Boc)-α-methylalanine (BMA) and N-t-Boc-α-methylalanine methyl ester (BMAM) in fast atom bombardment mass spectrometry (FAB-MS) were examined. Fragmentation and structural features of the [M+H]⁺ and [M+Li]⁺ ions are interpreted by using tandem mass spectrometry (MS/MS). The order of the activity of dissociation reaction [M+H]⁺[M+Li]⁺ is suggested from the relative peak intensities in the FAB mass spectra, I[M+H-fragment]⁺/I[M+H]⁺ and I[M+Li-fragment]⁺/I[M+Li]⁺. Furthermore, MNDO optimized structures of the [M+H]⁺ and [M+Li]⁺ ions are compared with the structures of the [M+H]⁺ and [M+Li]⁺ ions proposed by FAB-MS.

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A new application of tandem mass spectrometry to experimental studies of the fragmentation pathway is described. Metastable ion spectra of the naturally occurring isotope peaks as well as collision-activated dissociation spectra can give us a unique information on the ions. Time averaging of the spectra improves the spectral quality.

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The mechanism and reduction methods for the formation of interfering ion species in inductively coupled plasma mass spectrometer were described. The interfering ion species were classified into three groups according to the formation mechanism; (1) polyatomic ions due to argon and solvent, (2) refractory monoxides and hydroxides, and (3) doubly charged ions. Polyatomic ions due to argon seem to be formed by the collision-induced reactions of atom with ion in the micro plasma in the interface region between the sampling and skimmer cones (interface plasma), not in the ICP because their dissociation energies are quite small (below 2 eV except for ArH⁺) and most of polyatomic ions were dissociated into atom and ion in the ICP. Thus, the lowering of the plasma temperature and/or the secondary discharge occurring at the tip of sampling cone led to less frequency of the collision-induced reactions, resulting in the reduction of formation of polyatomic ions. Refractory monoxides and hydroxides whose dissociation energies exceed 4 eV were stable even in the ICP. Monoxide ion to element ion ratio depends on the plasma temperature, the number density of oxygen in the ICP and the time required for the desolvation of analyte particles. The signal for doubly charged ion increased under the condition that the effect of the secondary discharge was large, which was eliminated by reducing the inductively coupling between the ICP and the load coil.

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