基質輔助雷射脫附游離法的機制及其應用

Mechanism of Matrix-Assisted Laser Desorption/Ionization (MALDI) and Its Applications

李竹平(Chuping Lee)

指導教授 ： 李遠哲

共同指導教授 ： 倪其焜

國立臺灣大學/理學院/化學研究所/博士(2018年)

https://doi.org/10.6342/NTU201800617

摘要

質譜儀是廣泛用於微量分析的技術，在各種游離方法中，基質輔助雷射脫附游離 法是一種“軟性”的游離法，而且特別適用於難揮發和易熱分解的生物大分子。雖 然基質輔助雷射脫附游離法已經發展了三十餘年，但目前尚無法證明其確切的游 離機制，因此我們欲釐清其機制，同時希望能克服在應用上遇到的難題。本論文 包含三個實驗，分別研究帶質子的離子的產生，帶金屬離子的產生，及比較各種 軟游離法。
首先研究蛋白質類的分子在基質輔助雷射脫附游離法中，如何產生帶質子的離子 的，在過去的研究中，起始離子的生成備受爭議，陸續有很多不同的模型被提出 來去解釋起始離子的生成機制。根據我們實驗室先前的研究，熱致質子轉移模型 很可能是主要的機制，在第一個實驗中，為了更進一步證明其可能性，分別將不 同質子親和力的胺基酸(精胺酸，組胺酸和甘胺酸)以不同濃度加入基質(2,4,6-三 羥基苯乙酮)中，偵測並統計其帶正離子訊號的總和(包含帶質子的基質離子和帶 質子的分析物離子)。以略高於閥值的雷射光通量偵測，加入 1%最大質子親和力 的精胺酸在基質中，會比純的基質提升約 55 倍的訊號，組胺酸次之，而甘胺酸 幾乎不會提升訊號，若使用較高的雷射光通量，其訊號大小並未明顯改變，實驗 的結果和熱致質子轉移模型的預測有相同的趨勢，與其他模型的預測有明顯不 同。
在過去大部份的研究中，都著重於蛋白質類的游離是如何形成帶質子的離子，很 少提到醣類的游離是如何形成帶鈉離子的醣類離子，雖然預形成離子模型並未有 足夠的證據，但仍是最常被提及的機制。在第二個實驗中，為了瞭解分析物形成 帶金屬離子的游離機制，我們藉由三個子實驗研究可能形成帶金屬離子的原因。 第一個子實驗在加入鹽類的基質(2,5-二羥基苯甲酸)溶液中製備單晶，比較同一 條件下得到的晶體，相較於用去離子水洗去表面可能殘存的鹽類的晶體，未洗過 的晶體有較大的金屬相關離子訊號，可得知金屬相關離子的形成主要來自於表面上的鹽類，並非存在於晶體缺陷中的預形成離子。第二個子實驗分別製備兩種樣 品，一種是將基質，鹽類和分析物配成混合溶液，待乾燥結晶後再磨成細粉。另 一種未使用溶劑，是直接將基質，鹽類和分析物磨成細粉末再進行混合，未使用 溶劑的樣品，不會經過共結晶的過程，依然可偵測到帶金屬離子的基質和帶金屬 離子的分析物。當增加研磨時間使其更均勻時，未使用溶劑的樣品會有更強的分 析物訊號，因此對於金屬相關離子的生成，樣品混合的均勻度比預先形成離子更 為重要，基質分子和鹽分子接觸好的話，可增加金屬相關離子的產生。在第三個 子實驗中，可觀察到帶質子和帶金屬離子在雷射光通量的波動下有正相關，但和 雷射的波動性無明顯關係，這項觀察說明產生質子和產生金屬離子的效率應是受 相同因子影響，因此我們提出熱致鹽解離反應，解釋金屬相關離子的生成。合併 熱致質子轉移反應和熱致鹽溶解反應成為熱致游離模型，可以同時解釋兩類不同 離子的生成。
在各種游離方法中，雖然基質輔助雷射脫附游離法是比較“軟性”的游離法，但應 用於帶有磺酸根和唾液酸的醣類時，仍然會在游離或脫附的過程中碎裂，以至於 無法偵測到完整離子或是其訊號很小。近年來有很多軟游離的相關研究如下:(1) 冷凍法，(2) 三層法， (3) HgTe 奈米材料， (4) 5-甲氧基水楊酸(MSA)， (5) 2,5- 二羥基苯甲酸丁胺(DHBB)和(6) 基質輔助游離法(MAI)。但上述的研究分別使用 不同的分析物，因此不同的方法無法相互比較，在第三個實驗中，我們用相同的 分析物(神經節苷脂 GD1a，肝素 I-S，聚麥芽三糖和右旋糖酐)，比較哪個軟游離 法能兼具訊號提升和避免分子碎裂的效果。實驗的結果顯示冷凍法能避免分析物 在游離過程中碎裂，增加完整結構的訊號，進而提升分析物離子訊號。

關鍵字

基質輔助雷射脫附游離法 ； 游離機制 ； 熱致質子轉移 ； 熱致鹽溶解 ； 熱致游離模型 ； 基質輔助游離法 ； 軟游離法 ； 冷凍法

並列摘要

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.

並列關鍵字

matrix-assisted laser desorption/ionization (MALDI) ； ionization mechanism ； thermal proton transfer ； thermally induced dissolution of salt ； thermal model ； matrix-assisted ionization (MAI) ； soft ionization ； frozen

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