蛋白質體學及應用2
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(2) Technology . Gel spot excision and digestion • Individual variant spots • Washing (destaining) • Digestion (trypsin) • Peptide extraction • Clean-up (desalting).
(3) High-throughput analysis . Robotics (1) • Gel-spot excision . . Driven from gel image Cuts out gel spots Transfers to microtitre plates.
(4) High-throughput analysis . Robotics (2) • Protein digestion . Washes gel pieces Digests with trypsin Extracts peptides Desalts peptides Applies peptides to MALDI plate.
(5) Technology . Protein identification • Mass spectrometry . MALDI/TOF-MS. . Q-Tof-MS/MS.
(6) Database Search (Bioinformatics) Amino AminoAcid Acid Analysis AnalysisData Data Mass MassSpectrometry Spectrometry Data Data. AA composition database Database Search. 1021 1386. 854 1524. N-Terminal N-TerminalSequence Sequence Analysis AnalysisData Data PI PIand andMW MW from from2-D 2-D. 600. Target Protein Identification. Experimental Data 2001 Proteomics Group, Institute of Biological Chemistry, Academia Sinica. 1600. sequence tag database. protein + genome sequence database.
(7) 利用其他分子生物技術配合分析: Cell mapping, and identification of proteins in complexes: 共同沈澱法 or "pull-down" techniques using antibodies directed against one of the component proteins. • Coprecipitation using affinity-tagged recombinant proteins and antibodies directed against the "tag" epitope • Protein-affinity-interaction chromatography (e.g., using recombinant glutathione Stransferase (GST)-fusion proteins and glutathione-affinity chromatography) • Isolation of intact multiprotein complexes (e.g., nuclear pore complexes, ribosome complexes, and spliceosomes)..
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(9) 比較蛋白體學 Comparative Proteomics . 利用不同螢光標定法可正確定量樣品 Fluroscence label (Cy3, Cy5).
(10) DiGE: Quantitative 2D-PAGE sensitivity = ~200pg -mgs . . Sample multiplexing: 由Minden group at Carnegie Mellon University in Pittsburg發表, 克服傳統蛋白體定量不準的 問題. 待比較的不同樣本事先以 (Cy3, Cy5) 處理, 之後混合後在同一片膠體中分析-Difference Gel Electrophoresis (DiGE)..
(11) DIA: Difference in gel analysis . . For a DIA analysis, samples are minimally labelled with either Cy3 or Cy5 fluorescent dyes, and then pooled prior to 2D PAGE. The same isoform with the different labels will co-migrate • Fluorescent ratios can be compared after normalization. • The reported ratio indicates changes in expression levels.. . A reciprocal gel is run where the dye label is reversed. • Avoid differences in reactivity between dyes for the proteins..
(12) . Ünlü,M., Morgan, M. E., and Minden, J. S. (1997). Difference gel electrophoresis: a single gel method for detecting changes in cell extracts. Electrophoresis,18, 2071-2077. Pierce.
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(14) Gel Image 2D Gel Separation. Excitation λ1 (550 nm). Excitation λ2 (649 nm). Image 1. Image 2 Image analysis. Protein identification.
(15) Gel Image 97000 66000 45000 30000 20100 14400. 3.2. 4.0. 5.0. 5.5. 6.0. 7.0 8.0 9.0 10.0. CNT1 labeled with Cy3. 3.2. 4.0. 5.0. 5.5. 6.0. 7.0 8.0 9.0 10.0. KDML105 labeled with Cy5.
(16) Combined 2 images. 97000 66000 45000 30000 20100 14400. 3.2. 4.0. 5.0. 5.5. 6.0. 7.0 8.0 9.0 10.0.
(17) Imaging of Fluorescently labelled 2D gels Fluorescently labelled gels are imaged using a Typhoon 9400 scanner.
(18) Sparking:Internal standard could be adopted Pooled internal standard label with Cy2. Cy2. Protein extract 1 label with Cy3. Cy3. Protein extract 2 label with Cy5 Mix labelled extracts. Cy5 2D separation. Image gel with Typhoon Variable Mode Imager. Image analysis and data quantitation with DeCyder Differential Analysis Software.
(19) 生物質譜 BIOLOGICAL MASS SPECTROMETRY . . i) 雷射輔助基質脫附游離法-飛行時間質譜 MALDI-TOF ii)電噴灑法-四極柱質譜 ESI-Q-Tof iii)液相電層分析-質譜 LC-MS iv) LC-MS/MS.
(20) . 自early 1990’s, 質譜儀的兩大發現 • 電噴灑游離法Electrospray ionisation (ESI) and雷射輔助基質脫附游離法 matrixassisted laser desorption/ionisation (MALDI) were developed by Fenn et al. (1989) and Karas and Hillenkamp (1988), respectively. • 軟式游離法 Soft ionisation: very little internal energy is imparted into the ions during ionisation, resulting in the formation of intact ions, with minimal fragmentation..
(21) MALDI-TOF mass spectrometry . 雷射輔助基質脫附游離法--Matrix-assisted laser desorption/ionizationtime of flight (MALDI-TOF) mass spectrometry • 適合分析蛋白等大分子, 應用範圍廣, 已發表如: • protein and nucleic acid sequence, structure, purity, heterogeneity, cleavage, post-translational modification, and a host of other molecular characteristics that are often difficult to study by other means.. . MALDI-TOF 也可用在 QC tool: • verify peptide, protein, and DNA syntheses, etc..
(22) MALDI 主要三步聚:. • 游離 Ionisation, • 依質量分離 Mass separation, and • 偵測 Detection.. . . MALDI-TOF: 游離結果大部分帶一價電 i.e. (M+H)+, where M = the biological molecule and H = H+ (or a proton), 分子不會破碎. 離子由雷射激發 • Sample mixed with matrix • α-cyano-4-hydroxycinnamic acid: commonly used for peptide analysis • 2,5-dihydroxybenzoic acid (2,5 DHB) – sugar analysis • Sample: pico gram. . 基質可吸收雷射能量後轉移給待測分子. • laser (337 nm for N2 lasers) • A dense plume containing matrix and analyte molecules is produced and analyte molecules interact with hydrogen atoms from the matrix to form mainly singly charged (M+H)+ ions..
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(24) . . . 離子經TOF裝置後,由 microchannel plate detector (MCP)偵測. 反轉裝置 Reflection:a uniform electric field is generated at the end of the TOF tube which effectively pushes the ions back in the opposite direction.—可增加飛行時間. The mass range of a TOF analyser is, theoretically, infinite although, practically, it has an upper mass range of 750 kDa in linear mode and 100 kDa in reflectron mode..
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(26) PROTEIN IDENTIFICATION BY PEPTIDE MASS MAPPING • COMPUTER EXERCISE #3: Investigate peptide mass mapping used for protein identification.
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(29) Electrospray QuadrupoleTime-of-Flight (ESI-Q-Tof) . . ESI-MS was first reported in 1968 by Dole et al. improved at 1984 (Yamashita and Fenn, 1994). 可串接在液相層析儀之後,目前最微量的 nanoES (Wilm and Mann (1994, 1996), nanoelectrospray) 使用 gold tipped glass capillaries. (<50 fmoles of total protein) 20-50 nL min-1..
(30) . . 電噴灑法毛細管尖端使用高電壓 ( ~ 3-4 kV), 導致形成極細的帶電液滴, 內含離子(ions of the type (M+nH)n+, where M = the peptide molecule, nH is the number of protons attached to the molecule and n+ is the net charge of the biological ion.) 經由揮發後, 去除水份後分子帶電並進入分析儀 The multiply charged gas-phase ions are then formed as a result of desorption processes which occur due to evaporation of the solvent droplets (Iribarne and Thompson, 1976)..
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(32) LC –MS/MS.
(33) . ESI 與 MALDI不同點: • •. . 形成一系列帶不同電荷的離子. 分子在液相中分離帶電.. 可偵測高分子量的分子: • Relatively high molecular weight samples can be analysed by mass spectrometers with modest mass ranges (because mass spectrometry is concerned with the measurement of mass-to-charge (m/z) ratio, as opposed to mass)..
(34) . ESI可串接不同的質譜裝置,如: • quadrupoles, quadrupole ion traps, quadrupole time-of-flight (Q-tof) hybrid instruments and time-of-flight.. . 可使用串接成 tandem mass spectrometry or MS/MS, 可做定序並使 用極少量的分子即可完成分析..
(35) MALDI vs ESI.
(36) MS/MS . 質譜指紋比對 • 利用蛋白切除之小片段比對資料庫可得到吻合的蛋白 • first developed by several groups in 1993 (Henzel et al., Mann et al., Pappin et al., Yates et al.).. . 指紋比對的缺點 • (a) 某些蛋白在資料庫中沒有完整資料 • (b) 指紋資料可能由多個蛋白混合 (the map represents a mixture of proteins).. . MS/MS: Mann and Wilm (1994) and Eng et al. (1994), peptide sequencing techniques using which compared database peptide sequences with MS/MS data..
(37) 利用 MS/MS-TOF 做蛋白質定序 • 小片段 peptides 利用低能撞擊(low-energy collision-induced dissociation (CID) processes)使其分解. • During low energy collisions in the 四極 柱 (Q-Tof)或其他MS, y type ions最易出現 (C-N bond) (retention of the charge at the C-terminal side) and some low molecular weight b type ions (retention of the charge at the N-terminal side)..
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(41) ESI-MS/MS spectrum of a doubly charged ion (m/z 523.29) of a trypsin autolylsis product from porcine trypsin. Subtraction of the masses of adjacent fragment ion peaks (y-type) corresponds to the masses of the amino acids in the peptide chain. Hence, the complete sequence of the peptide is LSSPATLNSR..
(42) Posttranslational modification: • More than 200 kinds of Posttranslational modifications (PTMs). . e.g., methylation: 14.0269 Mw, GalN: 161.1577,phosphorylation: 79.9799 Mw, etc.. • Two major PTMs of proteins: phosphorylation, and glycosylation. . 可利以MS/MS等質譜方法決定後修飾位置..
(43) 利用質譜儀偵測蛋白質修飾(e.g., Phosphorylation detecting by MS) MALDI:(example-利用PSD法) 1. Post source decay (PSD): perform PSD on reflectron-equipped MALDI-TOF at first field free region. . P-Ser/Thr: loss of H3PO4 -- 98 Dalton. P-Tyr: loss of HPO3 -- 80 Dalton. •. Annan and Carr, 1996. 2. Alkaline phosphate treatment: •. MALDI before and after alkaline phosphate treatment..
(44) Linear Mode. 98Da. PSD, then reflectron 80Da.
(45) 蛋白質晶片 (Protein Microarray) Poor coorelation between mRNA and protein expression levels. DNA chips fail on PTMs signals. Applications: • Study enzyme-substrate, DNA-protein, protein-protein interactions on proteomic scale. • MacBeath and Schreiber (2000): protein arrays containing more than 10,000 proteins..
(46) Classes of capture molecules for protein microarrays..
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(49) . http://www.ciphergen.com.
(50) 蛋白質體網站 . Expasy (http://tw.expasy.org) etc….
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(53) http://proteome.sinica.edu.tw/pro_technology.asp.
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