陳仲瑄Chen, Chung-Hsuan臺灣大學:化學研究所張瑜玲Chang, Yu-LingYu-LingChang2010-06-302018-07-102010-06-302018-07-102009U0001-3107200900561900http://ntur.lib.ntu.edu.tw//handle/246246/187548在質譜儀的應用上，基質輔助雷射脫附游離法及電噴灑游離法是現階段常用來研究蛋白質或核酸等生物分子的分析工具。此外，由於生物科技的快速發展，醣類分子在生物體內的重要性也逐漸受到重視。研究發現，細胞與細胞間的辨識作用，蛋白質功能的正確與否，以及許多與代謝相關的疾病，都受其表面修飾之醣分子所影響。發展快速且正確分析醣類分子的方法越見重要性。但由於醣類本身質子吸附能力較蛋白質或核酸等生物分子來的低，導致醣類在質譜儀中離子化效率較差，增加偵測醣類分子的困難性。 為了增進醣類分子在質譜儀中的偵測效率，透過還原胺化的方式，於寡醣分子Maltoheptose (DP7)上修飾胜肽片段Bradykinin，利用質子吸附能力較高的胜肽片段提高醣類的游離效率。發現在基質輔助雷射脫附游離質譜儀中可增強訊號強度，同時偵測極限可由原先 54.6±14.9 fmol降低至 1.2±0.6 fmol；而在電噴灑質譜儀中則是可增強訊號強度最多8倍。 另一方面，也是利用還原胺化的方式，在寡醣分子DP7上修飾2-amino-1-naphthalensulfonic acid (ANS)或7-amino-1,3-naphthalendisulfonic acid (A13S)。在基質輔助雷射脫附游離質譜儀之負電模式下，藉由ANS / A13S易失去氫離子而帶負電的特性，使原先在負電下無法偵測之醣類分子，偵測極限可達 3.9±1.6 fmol / 2.7±0.9 fmol，此實驗數據優於前人所發表之60fmol；而在電噴灑質譜儀中，DP7-ANS與DP7-A13S之訊號強度最多可為DP7之9倍與13倍。 此外，研究發現，以離子液體DHBB (2,5-dihydroxybenzoic acid butylamine)為基質輔助雷射脫附游離法之基質使用，可偵測大的醣類分子。 將此觀念延伸至電噴灑游離質譜儀中，使用DHBB或CHCA-B (α-cyano-4-hydroxycinnamic acids butylamine)兩種離子液體進行分析多醣分子Pullulans (PL6K / 10K)之實驗。發現加入離子液體後，可改善醣類分子之訊雜比並增強訊號強度。分析圖譜可知，在正電模式下偵測時，正電荷由離子液體中之[butylamine+H]+所提供；同理在負電模式下，帶負電的[DHB-H]-或[CHCA-H]-也會吸附並增加醣類分子之游離效率；且電荷之來源完全由離子液體所提供，簡化醣類分子因鈉離子或鉀離子吸附而帶電的可能性，幫助回推多醣分子之分子量。 本實驗透過兩種不同的策略：針對醣類分子修飾胜肽片段或是特定基團，及將離子液體應用於電噴灑質譜儀中，皆可增加醣類在質譜儀的偵測效率，降低偵測極限或增強訊號強度，改善訊雜比。Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) were successfully developed as the soft ionization techniques for biomolecules, such as proteins and nucleotides. The benefit of MALDI-mass spectrometry (MS) for analysis of polymers is predominantly from the formation of singly charged molecular ions that lead to simple mass spectra for polymer distribution analysis. In view of the increasing biological importance of saccharides associated with cell-cell recognition, protein targeting and metabolic diseases, oligosaccharides and their glycoconjugate research attracted more attention lately. But the ionization efficiency of saccharides is low compared with other biomolecules due to the low proton affinity of carbohydrates. In order to detect oligosaccharides, we utilized the reductive amination processes to produce glycopeptides (DP7-BK) and derivatives of oligosaccharides with 2-amino-1-naphthalensulfonic acid (DP7-ANS) or 7-amino-1, 3-naphthalendisulfonic acid (DP7-A13S). In our study, modifications were found to improve the detection limit of MALDI mass spectrometry. In MALDI-MS, the detection limit of DP7-BK 1~9 is 1.2±0.6 fmol which is better than 54.6±14.9 fmol of DP7. The detection limits of DP7-ANS and DP7-A13S are 3.9±1.6 fmol and 2.7±0.9 fmol in negative ion mode. In ESI-MS, the signal intensity of DP7-BK 1~9 is 8 times higher than that of DP7 and the signal intensities of DP7-ANS and DP7-A13S in negative ion mode are 9 times and 13 times higher than that of DP7, respectively. Ionic liquid (IL) has been used as the matrix to increase ionization efficiency of polysaccharides in MALDI-MS. In this work, we applied IL in ESI-MS. The ionic liquid used are 2, 5-dihydroxybenzoic acid butylamine (DHBB) and α-cyano-4-hydroxycinnamic acids butylamine (CHCA-B). The signal to noise ratios (S / N) of polysaccharides PL6K / 10K are better in positive ion mode. Ion attachments from ionic liquid to polysaccharides are observed. This is the first time that addition of ionic compounds allows us to predict ionic adducts in ESI-MS.Contentsbstract in Chinese……………………………………………………………………. 2bstract………………………………………………………………………………... 4hapter 1. Introduction…………………………………………………………………...16. The background of matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS)…………………………………………………...19. History of MALDI-TOF…………………………………..………………21. The sample preparation and the matrices of MALDI……………………..22. Ion formation mechanisms in MALDI……………………………………26. Time-of-flight mass analyzer……………………………………………...31. The background of electrospray ionization mass spectrometry (ESI-MS)…...36. History of ESI-MS………………...………………………………………37. The principle and mechanism of ESI……………………………………..39. Ion trap mass analyzer…………………………………………………….42. The modification of oligosaccharides…………………………………………44. Peptides modification…………………………...………………………...44. Aromatic compounds modification……………………….……………....45. Ionic liquid (IL)……………………………………………………………….48. The application in MALDI-MS…………………………………………..48. Ionic liquid in ESI-MS………...…………………………………………. 50. The purpose of the thesis……………………………………………………...51hapter 2: Experimental methods and materials . Experimental samples. The reagents to modify oligosaccharides: peptides and aromatic compounds………………………………………………………………..53. Reductive amination………………………………………………………54. Polysaccharides and ionic liquid………….……………………………... 55. MALDI-TOF MS. Instrumentation…………………………………………………………...58. Matrix preparation………………………………………….……………. 58. The standard of mass calibration………………………………………….59. ESI - ion trap MS. Instrumentation…………………………………………………………...60. The sample preparation…………………………………………………...60hapter 3: Results and discussion. Modified oligosaccharides in MALDI-MS. Oligosaccharides were attached to peptides1) The detection limit for various matrices……………………...…….. 612) The effects of peptides length and amino acid sequences…………... 64. The oligosaccharides are attached to amino-1-naphthalensulfonic acid….70. Modified oligosaccharides in ESI-MS. Oligosaccharides were attached to peptide sequences1) The results of specific target mass setting (Target Mass: 1150Da)….752) The results of non-specific target mass setting………………………81. Oligosaccharides are attached to amino-1-naphthalensulfonic acid 1) DP7-ANS (2-amino-1-naphthalensulfonic acid, M.W=1358Da).........862) DP7-A13S (7-amino-1,3-naphthalendisulfonic acid, M.W=1438Da)..89. The application of ionic liquid for carbohydrates analysis in ESI-MS. DP7 (M.W=1153Da) was analyzed preliminaries in positive mode1) 1nmol/μl DHBB addition……………………………………..……..932) 5nmol/μl CHCA-B addition………………………………………....95. Ionic liquid (IL) are applied to Pullulans whose average molecular weights are 5900Da (PL6K) and 9600Da (PL10K).1) The detection in positive mode…………………………………….962) The detection in negative mode…………………………………...100hapter 4: Conclusions……………………………………………………………....104eferences…………………………………………………………………………...106ables………………………………………………………………………………..109igure contentsigure 1: The structure of mass spectrometry…………………………………….........20igure 2: A schematic diagram of the mechanism of MALDI………………………...23igure 3: The two-step model of ion formation in UV-MALDI………………………27igure 4: The pooling reactions of matrix excited states……………………………....28igure 5: Example of the time-depended evolution of a MALDI sample in the photoionization / pooling model…………………………………………….29igure 6: The sketch of the major processes proposed in cluster models of MALDI…30igure 7: The linear / reflectrons time-of-flight mass analyzer………………………...34igure 8: A schematic of the mechanism of ion formation in ESI…………………….39igure 9: The droplets shrink during electrospray ionization process…………………40igure 10: The ion trap mass analyzer…………………………………………………42igure 11: The unit maltotriose of polysaccharides pullulans…………………………56igure 12: IL α-cyano-4-hydroxycinnamic acid butylamine salt (CHCA-B)…………57igure 13: IL 2, 5-dihydroxybenzoic acid butylamine (DHBB)……………………....57igure 14: The detection limit of DP7 and DP7-BK 1~9, the matrix is THAP/DHB....63igure 15: The mixture of DP7 and DP-BK, THAP as the matrix…………………….65igure 16: The mixture of DP7 and DP-BK, CHCA as the matrix……………………67igure 17: The mixture of DP7 and DP-BK, DHB as the matrix……………………...68igure 18: The detection limit of DP7-ANS and DP7-A13S, DHB as the matrix……..72igure 19: The detection limit of DP7-ANS and DP7-A13S, 3-AQ as the matrix…….73igure 20: The structure of 3-aminoquinoline (3-AQ)…………………………………73igure 21: The mixture of DP7 and DP-BK in ESI-MS, 100fmol/μl…………………..77igure 22: The mixture of DP7 and DP-BK in ESI-MS, 250fmol/μl…………………..78igure 23: The mixture of DP7 and DP-BK in ESI-MS, 500fmol/μl…………………..79igure 24: The mixture of DP7 and DP-BK in ESI-MS, 1pmol/μl…………………….80igure 25: The sum of DP7-BK 1~9 signal intensity is 3.14 times higher than DP7, 100fmol/μl……………………………………………………………….....82igure 26: The sum of DP7-BK 1~9 signal intensity is 3.68 times higher than DP7, 250fmol/μl………………………………………………………………….83igure 27: The sum of DP7-BK 1~9 signal intensity is 4.25 times higher than DP7, 500fmol/μl………………………………………………………………….84igure 28: The sum of DP7-BK 1~9 signal intensity is 4.75 times higher than DP7, 1pmol/μl…………………………………………………………………....85igure 29: The mixture of DP7 and DP7-ANS in ESI-MS, the target mass setting is 1150Da, 100fmol/μl ~ 5pmol/μl……………………………………………87igure 30: The signal intensity of DP7 and DP7-ANS for 100fmol/μl ~ 5pmol/μl……88igure 31: The mixture of DP7 and DP7-A13S in ESI-MS, the target mass setting is 1150Da, 100fmol/μl ~ 5pmol/μl……………………………………………90igure 32: The signal intensity of DP7 and DP7-A13S for 100fmol/μl ~ 5pmol/μl…..91igure 33: DP7 (upper) and the addition of 1nmol/μl DHBB (down) in ESI-MS….. ...94igure 34: DP7 (upper) and the addition of 5nmol/μl CHCA-B (down) in ESI-MS…..95igure 35: PL6K (upper), PL6K + DHBB (middle) and PL6K + CHCA-B (down) in positive mode for ESI-MS………………………………………………….97igure 36: PL10K (upper), PL10K + DHBB (middle) and PL10K + CHCA-B (down) in positive mode for ESI-MS……………………………………………….98igure 37: PL6K (upper), PL6K + DHBB (middle) and PL6K + CHCA-B (down) in negative mode for ESI-MS………………………………………………..100igure 38: PL10K (upper), PL10K + DHBB (middle) and PL10K + CHCA-B (down) in negative mode for ESI-MS……………………………………………..101able contentsable 1: The structures of different matrices…………………………………………109able 2: Peptide or aromatic compounds are attached to DP7……………………….110able 3: The detection limit of DP7, DP7-BK 1~9, DP7-ANS and DP7-A13S for different matrices…………………………………………………………....111able 4: The m/z of DP7 and DP-BK 1~3, DP7-BK 1~5, DP7-BK 1~7, DP7-BK 2~9 and DP7-BK 1~9 in ESI-MS……………………………………………….. 1121692036 bytesapplication/pdfen-US基質輔助雷射脫附游離質譜儀電噴灑游離質譜儀離子液體醣類偵測極限醣類的修飾MALDI-MSESI-MSionic liquid (IL)detection limit of oligosaccharidesglycopeptides2-amino-1-naphthalensulfonic acid7-amino-1,3-naphthalendisulfonic acidthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/187548/1/ntu-98-R96223212-1.pdf