李遠哲臺灣大學：化學研究所廖幸芝Liau, Shing-ChihShing-ChihLiau2007-11-262018-07-102007-11-262018-07-102006http://ntur.lib.ntu.edu.tw//handle/246246/51930本篇論文主要工作是運用傅立葉轉換離子迴旋共振質譜儀質譜儀(FTICR)配合紅外雷射來研究蛋白質中的非共價鍵能以及多肽鏈以非共價鍵方式修飾奈米鑽石表面應用於增強DNA在介質輔助雷射脫附游離飛行式質譜儀(MADLI-TOF MS)中的偵測。 第一部份： 實驗利用傅立葉轉換離子迴旋共振質譜儀（FTICR）以及電灑（ESI）離子化方式,結合脈衝式3μM可調式OPO laser(Optical Parametric Oscillator), 藉由紅外多光子解離法(IRMPD)量測蛋白質非共價鍵的活化能,作為研究蛋白質非共價鍵能量的方法。 肌血紅蛋白（holomyoglobin）含有一個非共價鍵結的血基質(heme）藏在此蛋白質中, 血基質的中心有一個鐵配位,此血基質的鐵離子有兩種不同氧化態：[Fe(II)-Heme]和[Fe(III)-Heme]。先前已有人觀察到[Fe(II)-Heme]與aMbn+鍵結活化能較[Fe(III)-Heme]+與aMb(n-1)+強,但是[Fe(II)-Heme]與aMbn+絕對能量尚未被量測出來。 首先將hMb帶有中心鐵為三價的[Fe(III)-Heme],利用ESI來產生多價數肌血紅蛋白離子[hMb]n+,將[hMb]n+離子隔離在FTICR的腔體中,利用OPO laser在3333cm-1照射,可產生不帶有血基質的apomyoglobin[aMb](n-1)+的離子碎片。此碎片來自於失去帶有+1價的血基質（碎片為[Fe(III)-Heme]+和[aMb](n-1)+）。藉由改變Laser強度以及照射時間,量測解離的碎片含量,和先前文獻量測0.9 eV來得到IRMPD方程式 的校正值s。 進一步將hMb還原,使同時含有中心鐵為二價的[Fe(II)-Heme]還原態和中心鐵為三價的[Fe(III)-Heme]+氧化態,利用相同方法產生離子並照射,可同時產生兩種不帶血基質的[aMb]n+和[aMb](n-1)+離子碎片分別來自還原態和氧化態。同上,改變Laser強度和照射時間,同時量測兩種解離碎片含量,觀測hMb n+ (n=9,10)得知[Fe(II)-Heme]與aMbn+的鍵結活化能較[Fe(III)-Heme]+與aMb(n-1)+高25%, 測得其相對能量,運用IRMPD方程式,即得其絕對鍵結活化能分別約為1.1 eV,0.9 eV。 第二部分： 介質輔助雷射脫附游離飛行式質譜儀(MADLI-TOF MS)是一種有效分析巨型生物分子例如蛋白質和去氧核醣核酸(DNA)的工具。但是因為其低離子化的效率和容易使DNA過分碎解,所以此技術的整體表現在DNA比蛋白質差。早期利用介質輔助雷射脫附游離飛行式質譜儀觀察DNA,主要集中在選擇不同基質和改善樣品的沉積方式。近期有較大的影響的是降低偵測極限和增加質量測量的正確性,此兩者對於低濃度的樣品偵測和分析複合物是很重要的幫助。 因此在此實驗,我們利用聚賴氨酸非共價鍵修飾後的奈米鑽石當作固相萃取。此奈米鑽石可以純化,濃縮,利用酵素分解DNA應用於介質輔助雷射脫附游離飛行式質譜儀。由實驗的結果顯示,利用此奈米鑽石固相萃取可以將極稀薄濃度的DNA溶液中濃縮增強質譜的訊號,也可從含有蛋白質或含有汙染物的溶液中藉由吸附分離出DNA。除此之外,酵素分解DNA可以在此奈米鑽石上進行,若是在溶液中進行DNA酵素分解,分解後的碎片也可以藉由此奈米鑽石來增強訊號,提高質譜儀的靈敏度。在此實驗所有的圖譜分析中,此法不會造成訊號被拓寬和質量發生位移。此聚賴氨酸非共價鍵修飾後的奈米鑽石粒子藉由靜電力與DNA形成穩定的複合物,單純利用去離子水清洗是無法將DNA洗去,我們可以用氨水和升溫將DNA從奈米鑽石的表面洗出。除此之外,在接下來的介質輔助雷射脫附游離飛行式質譜儀分析中,不需要事先分離聚賴氨酸或奈米鑽石。The thesis consists of two parts. In part I, we study dissociation of noncovalently bound heme from gaseous myoglobin ions by IRMPD with FT-ICR mass spectrometetry. In part II, we noncovalently coat polylysine on diamond nanocrystals for MALDI-TOF mass analysis of DNA oligoncleotides. The abstracts of both parts are shown below. PART I: We have studied detachment of heme prosthetic groups from gaseous myoglobin ions by infrared multiphoton dissociation (IRMPD) by means of Fourier- transform ion cyclotron resonance (FTICR) mass spectrometry. Multiply charged holomyoglobin ions (hMbn+) were generated by electrospray ionization (ESI) and transferred to an ICR cell, where the ions of interest were isolated and fragmented by irradiation with 3-μm photons, producing apomyoglobin ions (aMbn+). Both of oxidized and reduced forms of myoglobin (Fe (III)-hMbn+ and Fe (II)-hMbn+) were invistegated First, charged heme loss (with [Fe (III)-heme]+ and aMb(n-1)+ as the products) was detected for hMbn+ produced from a pure ferric myoglobin solution with different laser irradiation times. Next, both charged heme loss (with [Fe (III)-heme]+ and aMb(n-1)+ as the products) and neutral heme loss (with [Fe (II)-heme] and aMbn+ as the products) were detected concurrently for hMbn+ produced from a myoglobin solution pretreated with reducing reagents. The activation energies were determined by fitting these sets of experimental data separately to . With reference to Ea = 0.9 eV of charged heme loss for ferric hMbn+ determined by blackbody infrared radiation, a dissociation activation energy of 1.1 eV was deduced for the neutral heme loss of ferrous hMbn+ with n = 9 and 10.The difference in dissociation activation energy between these two channels is small, only ~25% (or 6 kcal*mol-1). PARTII: In this experiment, the amine-terminated diamond nano- crystals (~100 nm in diameter) were prepared by noncovalent coating of carboxylated/ ox- idized diamonds with poly-L-lysines (PL), which form stable complexes with DNA oligonucleotides in solution. While the complexes are sufficiently stable to sustain repeated washing with deionized water, the DNA molecules can be readily eluted after incubation of the diamond adducts in aqueous ammonium hydroxide at elevated temperatures. No preseparation of PL or diamond nanocrystals is required for subsequent MALDI-TOF mass analysis. A protocol based on amine-terminated diamond nanocrystals could be developed to isolate, concentrate, purify and digest DNA oligonucleotides in one pot for matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). It is shown that use of diamond nanocrystals as a solid-phase extraction support not only permits concentration of oligonucleotides in dilute solutions but also facilitates isolation of oligonucleotides from proteins in mixed solutions. Additionally, enzymatic digestions can be conducted on particle and, furthermore, the digests can be easily recovered from the solution to obtain base sequence information.論文摘要 5 Abstract 8 Part I Dissociation of Heme from Gaseous Myoglobin Ions by IRMPD with FT-ICR Mass Spectrometry Chapter 1 Introduction 11 1.1 Myoglobin 12 1.1.1 Myoglobin undergoes a structural change on bonding oxygen 16 1.2 The Heme 19 1.2.1 The Structure and Function of Heme 20 1.2.2 Spin state of the Center Iron of Heme 22 1.3 Myoglobin in the Mass Spectrometry 25 Reference 26 Chapter 2 Experimental Apparatus and Methods 27 2.1 Fourier Transform Ion Cyclotron Resonance Mass Spectrometry 28 2.1.1 FT ICR Theory 29 2.1.2 Fourier Transform Spectrometry 31 2.2 Electrospray Ionization: Ion Source 32 2.3 The activation methods with FTICR 34 2.3.1 Collision-Induced Dissociation 34 2.3.2 Blackbody Infrared Dissociation 36 2.3.2 IR Multiphoton Dissociation 38 2.4 The Experimental Setup 40 2.5 Prepare Myoglobin Solution 43 Reference 44 Chapter 3 Activation Energies for Dissociation of Ferric and Ferrous Myoglobin Ions Simultaneously from IRMPD 46 3.1 Previous Studies 48 3.1.1 Blackbody Infrared Dissociation 48 3.1.2 IR Multiphoton Dissociation Studies 50 3.2 The study of Myoglobin by IRMPD 53 3.3 Experimental Results and Analysis 55 Reference: 64 Chapter 4 Conclusion 66 Part II Polylysine-Coated Diamond Nanocrystals for MALDI-TOF Mass Analysis of DNA Oligonucleotides Chapter 1 Introduction 69 Chapter 2 Experimental Method 72 2.1 Chemicals and Materials 73 2.2 PL-coated diamond nanocrystals 73 2.2.1 Surface Functionalization 73 2.2.2 IR Measurements 74 2.3 DNA adsorption 75 2.4 Concentration and Extraction 75 2.5 Enzymatic digestion 76 2.6 MADLI-TOF MS 76 Chapter 3 Experiment Results and Discussion 78 3.1 Adsorption and elution of DNA from PL-coated diamond surfaces 79 3.2 Mass Analysis of Dilute DNA Solutions with Contaminants 81 3.3 Selective Extraction of DNA from Solutions Containing Proteins 83 References 95 Chapter4 Conclusion 971586665 bytesapplication/pdfen-US質譜肌血紅蛋白奈米鑽石Mass SpectrometryFT-ICRMALDI TOFnanodiamondmyoglobinthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/51930/1/ntu-95-R93223078-1.pdf