# Vacuum Ultraviolet Single-Photon Positionization of Amino Acids
## 3 Results
### (a) VUV photon number
The signals at _m/z_ = 168 and 153 represent the parent and fragment ions of the desorbed neutral THAP molecules (m = 168) ionized by VUV photons. The high ion intensity obtained from VUV-SP-positionization indicates the high sensitivity of VUV-SP-positionization compared with that of UV-MALDI. For 5% glycine in THAP, almost no ions related to glycine were observed in UV-MALDI (green line in Figure 4a). However, high ion intensities were discovered at _m/z_ = 75 and 30, corresponding to the glycine parent ion Gly\({}^{+}\) and fragment ion [Gly-COOH]\({}^{+}\), respectively, when VUV-SP-positionization was employed (blue line in Figure 4a). These signals represent desorbed neutral glycine molecules generated through UV laser desorption and ionized through VUV-SP-positionization. Ion signals from VUV-SP-positionization increased with an increase in the UV laser fluence (to 110 J/m\({}^{2}\)) because numerous neutrals were desorbed by the UV laser (Figure 4b). The mass spectra obtained when tryptophan was used as an analyte in THAP are presented in Figure 4c. Similar to Figure 4a,b, large THAP ion signals were generated through VUV-SP-positionization. Numerous THAP ions were generated, which could suppress the detection efficiency of a microchannel plate (MCP) detector for the ions arriving subsequently. Because tryptophan ions have a large molecular weight and arrive at the detector after THAP ions, a gated MCP was used to overcome suppression effects. The voltage applied to the MCP detector was lowered during the time period in which THAP ions were arriving. The voltage was then increased to its initial setting rapidly after this period to detect the ions that arrived after the THAP ions. Figure 4c shows the VUV-SP-positionization spectrum of tryptophan when the gated MCP was employed. The major signals related to tryptophan ions are those at _m/z_ = 204 and 130, corresponding to the tryptophan parent ion, Trp\({}^{+}\), and fragment ion, [Trp-NH\({}_{2}\)CHCOOH]\({}^{+}\), respectively. The mass spectra of 20 common amino acids were obtained using the same technique. Table 1 presents the fragmentation of each amino acid observed in the VUV-SP-positionization spectra. The fragmentations of all the amino acids, apart from arginine, were generally similar to those that would by obtained using electron impact (EI) ionization at 70 eV [63]. The dominant fragmentations of the amino acids were cleavages of the R-COOH bond and bond between C\({}_{\alpha}\) and C\({}_{\beta}\). However, the degree of fragmentation observed from VUV-SP-positionization was considerably less than that after EI ionization. The relative ion abundance obtained using UV-MALDI and VUV-SP-positionization was defined as follows:  
\[\text{Relative ion abundance}\ =\ \frac{\text{ion intensity of parent or fragment from VUV SPI}}{\text{ion intensity of AH}^{+}}\ \text{from UV}-\text{MALDI}\]  
The relative ion abundances are listed in Table 1 for a laser fluence of 110 J/m\({}^{2}\). For example, the ion intensity of the tryptophan fragment corresponding to _m/z_ = 130 obtained through VUV-SP-positionization was approximately 3600 times larger than that of the protonated tryptophan ions obtained through UV-MALDI, and the tryptophan parent ion intensity obtained through VUV-SP-positionization was 27 times larger than the protonated tryptophan intensity obtained through UV-MALDI. Protonated glycine was not observed in UV-MALDI. Relative ion abundance is the ratio of the ion intensity from VUV-SP-positionization to the noise level of UV-MALDI. The parent and fragment ions of aspartic acid and asparagine were not observed when THAP was used as a matrix; however, they were detected when the matrix was 2,5-DHB. The relative ion abundances of these 20 common amino acids indicate that the detection sensitivity of VUV-SP-positionization is generally 10-1000 times higher than that of conventional UV-MALDI.