多維高效液相層析線上連結質譜儀測定短胜肽胺基酸序列測定技術

Multi-dimensional high-performance liquid chromatography online coupled to mass spectrometer for short peptide amino acid sequence determination

廖健富(Chien-Fu Liao)

指導教授 ： 鄭建業

中原大學/理學院/化學研究所/碩士(2015年)

https://doi.org/10.6840/cycu201500830

摘要

本研究開發一種多維液相層析線上連結質譜儀系統，配合線上管柱轉換技術及線上核心切割取樣技術，發展短胜肽胺基酸序列定序方法，並應用於二胜肽阿斯巴甜與三胜肽穀胱甘肽之胺基酸序列定序。第一維親合掌性層析管柱可分離短胜肽電熱水解之胺基酸及胜肽產物，並以紫外光偵測；第一維分離出之胺基酸除半胱胺酸外可轉換至第二維配位子交換層析以螢光偵測器進行胺基酸鏡像體及胺基酸同分異構物的分離辨識，以了解胺基酸及其鏡像體種類;然後再將第一維層析管柱分離出之短胜肽與半胱胺酸等產物，依序轉換至另一第二維逆相層析管柱，以離子阱質譜儀測定半胱胺酸及各胜肽產物分子量與特性質譜碎片訊號。最後配合測得之胺基酸種類及其分子量，經由排列組合的方式，依照〝由小胜肽到大胜肽〞之順序組合定序出短胜肽之胺基酸序列。

本〝由下往上〞之胜肽胺基酸新穎定序方法已應用於阿斯巴甜(Aspartame)雙胜肽及穀胱甘肽(Glutathione)三胜肽之定序，發現阿斯巴甜是由L-天門冬胺酸及L-苯丙胺酸兩種胺基酸鏡像體所組成，穀胱甘肽是由甘胺酸、L-穀胺酸及L-半胱胺酸三種胺基酸鏡像體所組成。在離子阱質譜儀之正離子模式下的質譜偵測，發現到分子量m/z 281的[AspPhe+H]+二胜肽質譜訊號及氮端的羰陽離子m/z 116的[AspPhe-Phe]+質譜訊號，確定了阿斯巴甜的胺基酸序列為“天門冬胺酸-苯丙胺酸”，最後再經由嘗試錯誤方式，發現碳端苯丙胺酸修飾上甲氧基之m/z 180的 [AspPheOCH3-Asp+2H]+質譜訊號及m/z 295之[AspPheOCH3+H]+質譜訊號，證明了阿斯巴甜的結構為“天門冬胺酸-苯丙胺酸甲酯”。利用同樣方法先確定出穀胱甘肽由甘胺酸、L-穀胺酸及半胱胺酸三種胺基酸所組成，然後以質譜儀正離子模式下，找到雙胜肽氮端之羰陽離子質譜訊號，確定兩種雙胜肽分別為m/z 251的“穀胺酸-半胱胺酸”和m/z 179的“半胱胺酸-甘胺酸”，再藉由兩個雙胜肽所具有之共同半胱胺酸胺基酸殘基、氮端之羰陽離子m/z 130的[GluCysGly-Gly]+訊號、m/z 233的[GluCysGly-Gly]+訊號及m/z 308的[GlyCysGly+H]+訊號，確定三胜肽榖胱甘肽之“榖胺酸-半胱胺酸-甘胺酸” 序列。

螢光偵測器對L-天門冬胺酸、L-苯丙胺酸、甘胺酸及L-榖胺酸四種胺基酸偵測之偵測極限值範圍為0.03-0.28 μg mL-1。質譜儀偵測器對二胜肽“天門冬胺酸-苯丙胺酸”、“穀胺酸-半胱胺酸”及“半胱胺酸-甘胺酸”、阿斯巴甜、穀胱甘肽及L-半胱胺酸的偵測極限值範圍為1.04-2.46 ng mL-1。

關鍵字

多維高效液相層析 ； 配位子交換層析 ； 親和性層析 ； 逆相層析 ； 管柱轉換技術 ； 線上核心切割技術 ； 胺基酸鏡像體 ； 胺基酸序列定序 ； 甘胺酸 ； 穀胺酸 ； 半胱胺酸 ； 天門冬胺酸 ； 苯丙胺酸 ； 胜肽 ； 穀胱甘肽 ； 阿斯巴甜 ； 螢光偵測 ； 紫外光偵測 ； 電噴灑離子化離子阱質譜儀

並列摘要

Online column-switching technique and online heart-cutting sampling technique coupled multidimensional liquid chromatography mass spectrometric system was developed and used for the development of an amino acid sequencing method by using short peptides such as the dipeptide Aspartame and the tripeptide Glutathione. The first dimension column was an affinity chiral chromatographic column which was used for the separation of peptides and amino acids produced from the electric-thermal hydrolysis of short peptides and detected by an UV detector. Except cysteine, the amino acids separated by the first dimension chromatographic column were transferred to the second dimension ligand-exchange chromatographic column for further separation to learn about the kind of amino acid and the type of its enantiomer by a fluorescence detector. Then, the peptide products and cysteine separated by the first dimension chromatographic column were sequentially transferred to the other second dimension reversed-phase chromatographic column for further separation and detected by an ion-trap mass spectrometer to obtain the molecalur werght and characteristic mass fragment ion signal of various peptide products and cysteine. Finally, the amino acid sequence of the studied short peptide was determined in accordance with the order of "small peptide to large peptide" by matching the molecular weight of previously determined amino acids and the molecular weight information of peptide from mass spectrometry through the method of permutation and combination.

　　The novel "botom-up" amino acid sequencing method for short peptides has been used for determining the amino acid sequence of the dipeptide Aspartame and the tripeptide Glutathione. The determined amino acid enantiomers were L-aspartic acid and L-phenylalanine for Aspartame. The determined amino acid enantiomers were glycine, L-glutamic acid, and L-cysteine for Glutathione. In the positive ion detection mode of the ion-trap mass spectrometry, the molecular mass signal of dipeptide m/z 281 ([AspPhe+H]+) and the N-terminal carbo cation signal of m/z 116 ([AspPhe-phe]+) confirms that the amino acid sequence for Aspartame is "Aspartyl-phenylalanine (AspPhe)". Then, by trial-and-error, a methoxide functional group was modified on the C-termal phenylalanine to give a mass signal of m/z 180 ([AspPheOCH3-Asp+2H]+) and a mass signal of m/z 295 ([AspPheOCH3+H]+). Therefore, the structure of Aspartame is aspartyl phenylalamine methyl ester (AspPheOCH3). Similarly, the amino acid sequence of the tripeptide Glutathione was determined first by confirming the three constituents amino acid enantiomers, i.e. glycine, L-glutamic acid, and L-cysteine. Then, under the positive ion detection mode of mass spectrometer, the molecular mass signals of the two dipeptides m/z 251 ([GluCys+H]+) and m/z 179 ([CysGly+H]+) and their corresponding N-terminal carbon cation mass fragmentation signals were confirmend to suggest the tripeptide GluCysGly. Finally, the two N-terminal carbo cation mass fragmentation signals of m/z 130 ([GluCysGly-CysGly]+) and m/z 233 ([GluCysGly-Gly]+) and the tripeptide mass signal of m/z 308([GluCysGly+H]+) were identified to confirm the amino acid sequence of glutathione as "glutamyl-cysteinyl-glycine (GluCysGly)".

　　The limit of detection (LOD) of the fluorescence detector for the amino acid enantiomers of L-aspartic acid, L-phenylalanine, glycine, and L-glutamic acid were in the range of 0.03-0.28 μg mL-1; the LOD of the ion-trap mass spectrometer for the dipeptides "aspartyl-phenylalanine", "glutamyl-cysteine", and "cysteinyl-glycine", Aspartame, Glutathione, and L-cysteine were in the range of 1.04-2.46 ng mL-1.

並列關鍵字

Multi-dimensional high-performance liquid chromatography ； Ligand-exchange chromatography ； Affinity chiral chromatography ； Reversed-phase chromatography ； Column-switching technique ； Online Heart-cutting technique ； Amino acid enantiomers ； Amino acid Sequencing ； Glycine ； Glutamic acid ； Cysteine ； Aspartic acid ； Phenylalanine ； Peptide ； Glutathione ； Aspartame ； Fluorescence detection ； Ultraviolet detection ； Electrospray ionization-Ion Trap mass spectrometer

參考文獻

[64] 陳玉茹，Mass spectrometry and proteomics, 中華民國，南港，中央研究院。

[2] Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Robers, Peter Walter. Molecular biology of the cell, 4th ed, Garland Science, New York, 2002.

[3] Laylin K. James, Nobel Laureates in Chemistry 1901–1992, American Chemical Society, Chemical Heritage Foundation, Washington, DC, 1993.

[4] J. Kendrew, G. Bodo, H. Dintzis, R. Parrish, H. Wyckoff, D. Phillips, “Three-dimensional model of the myoglobin molecule obtained by x-ray analysis,”. Nature, 181, (4610), 662-666, 1958.

[6] J. Han, T. Ai, T. Nguyen,G. Li, “Chiral N-phosphonyl imine chemistry: asymmetric additions of ester enolates for the synthesis of β- amino acids.” Chemical Biology & Drug Design , 72, 120-126, 2008.