| Summary: | 博士 === 國立臺灣大學 === 化學研究所 === 106 === Matrix-assisted laser desorption/ionization (MALDI) is one of the important methods in mass analysis for biomolecules. To improve the application of MALDI in bioanalysis, it is vital to understand the ionization mechanism. Here we study the three topics, which are the mechanism of protonated ion, the mechanism of metal-related ion separately, and then we compare some softer ionization methods to find out the method soft enough for some fragile molecules in MALDI.
In the first topic of the protonated ion generation mechanism in MALDI, generating the first ions remains the most controversial part of the ionization mechanism. Several mechanisms have been proposed to explain the mechanism of ion generation in MALDI. However, the truthfulness of each mechanism in MALDI are difficult to determine because it is not easy to quantitatively measure the contributions of these mechanisms. The ionization mechanism of UV-MALDI was investigated by measuring the total cation intensity (not including sodiated and potasiated ions) as a function of analyte concentration (arginine, histidine, and glycine) in matrix of 2,4,6-trihydroxyacetophenone (THAP) using time-of-flight (TOF) mass spectrometer. The total ion intensity increased up to 55 times near the laser fluence threshold as the arginine concentration increased from 0% to 1%. The increscent were small for the case of histidine, and almost no increase occurred regarding for glycine. The increases became small for all analytes mentioned at high laser fluence. Here, thermal proton transfer model was used to predict the ion intensity as a function of analyte concentration. The increase of ion intensity can be explained by the thermal proton transfer model in the primary ion generation in MALDI.
In the second topic of the metal-related ion generation mechanism in MALDI, preformed ions are generally accepted as a unspoken mechanism. Three separate experiments were demonstrated that thermally induced dissolution of salts can make significant contribution in metal ion generation in MALDI. In the first experiment, ion intensities from two different types of samples were measured. Several single crystals were grown from the solution containing 2,5-DHB matrix and salt (LiCl and NaCl). One group of crystals was washed by deionized water before sending into mass spectrometer. The other group of crystals was used directly. The intensities of metal ion and metal adducts of the matrix ion obtained from unwashed crystals were higher than those from crystals washed with deionized water, indicating that metal ion and metal adducts of the matrix ion mainly generated from the surface, not inside the 2,5-DHB crystal. The contributions of preformed metal ions and metal adducts of the matrix ions inside the matrix crystals were minor.
In the second experiment, mass spectra of MALDI from two groups of samples were measured for comparison. One group of samples was the mixture of dried matrix powder, salt (LiCl and NaCl) and analyte powder. No solvent was used in this sample preparation method. The other group of samples was powder from the crystal of dried droplet, which the solution of droplet contained matrix, salt and analyte. Metal adducts of the matrix and analyte ion intensities generated from these two samples are similar, indicating that the contribution of the preformed metal adducts of the matrix and analyte ions were insignificant.
In the third experiment, the correlation between the metal-related ion intensity fluctuation and the protonated ion intensity fluctuation was observed, indicating that the generation mechanism of the metal-related ions is similar to that of the protonated ions. The thermally induced proton transfer model effectively describes the generation of the protonated ions; we suggest that metal-related ions are mainly generated from the salt dissolution in the matrix melted by the laser.
In the final topic, MALDI is one of the soft ionization methods in mass analysis for protein and glycan, but some fragile glycans with sulfate or sialic acid tend to lose the sulfate and sialic acid during the ionization process. The intensity of intact ion is usually small, and sometime no intact ions are observed at all. In recent years, many studies purposed new matrices to reduce the in-source and post-source decays as well as to increase the sensitivity of intact molecules. These matrices include (1) frozen sample (2) trilayer sample (3) HgTe nanostructures (4) 5-methoxysalicylic acid (MSA) (5) 2,5-dihydroxybenzoic acid butylamine (DHBB) (6) matrix-assisted ionization (MAI). Because different analytes were used in these studies, it is difficult to compare the softness of ionization for these matrices. In this study, we used these matrices and several fragile analytes to investigate softness of these matrices in the ionization process. These analytes include heparin disaccharide I-S, ganglioside GD1a, pullulan and dextran. We proved that frozen sample provide soft ionization and enhanced ion intensity.
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