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1、,Proteomics,Session 10 Mass spectrometry for proteomics,Mass sample preparation,General procedures for mass sample preparation,Washing staining material from the gelEquilibrium of gel to protease buffer conditionReduction and alkylation of proteinsDigestion of proteins to peptidesExtraction of pepti
2、desPurification of peptides (Optional),Prepare sample step by step,A. Spot picking將 1mL 藍色吸管尖端以手術刀切下約 0.5 mm。利用此吸管尖將 CBR 染色上有興趣的點取下，裝入1.5 mL 離心管。放入-20冰箱保存。,Prepare sample step by step,B. Washing and equilibration用1 mL ddH2O 洗膠體，每次10分鐘，洗兩次。加入1 mL 50mM NH4HCO3 / 50% acetonitrile（v/v）震盪15 分鐘，再去掉液體。加入0.
3、2 mL acetonitrile（gel會縮小變白），待其變白去掉 acetonitrile。加入0.5 mL 50mM NH4HCO3 ，震盪 5分鐘，再加入相同體積的acetontrile，震盪 15分鐘。去掉全部液體。加入0.2 mL acetonitrile，待膠體變白變小，去掉所有液體。打開離心管蓋，待膠體變乾（可以在離心管中彈跳）。,Prepare sample step by step,C. Reduction and alkylation加入50L 10mM DTT / 25mM NH4HCO3 （新鮮配置）於56 反應 45分鐘，去掉全部液體。加入50L 55mM iodo
4、acetamide（新鮮配置）於室溫避光30分鐘，去掉全部液體。加入0.2 mL 50mM NH4HCO3 /50% acetonitrile（v/v），震盪 15分鐘兩次，去掉全部液體。加入0.2 mL acetonitrile，待gel變白變小，移去所有的液體（越乾越好）。打開離心管蓋，待膠體變乾（可以在離心管中彈跳）。,Prepare sample step by step,D. Digestion加入1L trypsin 37 反應 30分鐘。再加入3L 25mM NH4HCO3 37 隔夜反應。,Prepare sample step by step,E. Extraction超音波
5、 (sonication) 震盪 10 分鐘 。加入3L ddH2O，超音波震盪 10分鐘後，將液體吸出放入新的離心管。舊管加入5L 50% Acetoneitrile / 1% Trifluroacetic acid（TFA），超音波震盪 10分鐘。重複一次。取出舊管的全部液體至剛剛的新管。 (For LC/MS/MS)抽真空，完全除去殘留液體。(For MALDI-Tof),Prepare sample step by step,F. Purification of peptide (for MALDI-Tof)材 料來自 E. 樣本 潤濕液（50：50, ACN：H2O, 0.1% TF
6、A）- 編號1 平衡與洗滌液（0.1% TFA in ddH2O）- 編號2 沖堤液（90：10, ACN：H2O, 0.1% TFA）- 編號3 Easytips (C18 resin) 標示廢液的離心管,Prepare sample step by step,F. Purification of peptide (for MALDI-Tof), continued步 驟 將少數樣品加入10L的編號 2。 將 P10 的吸量管設定至 10L，放置 Easytips 於 P10 的吸管上。 潤濕：吸取 10L編號 1 的溶液 3 次，拋棄每次吸取的溶液至廢液。 平衡：吸取 10L編號 2 的溶
7、液 4 次，拋棄每次吸取的溶液至廢液。 導入樣品：慢慢地吸取 10L的樣品，之後將樣品推出至離心管內；重覆此步驟 7 次。 洗滌：吸取 10L編號 2 的溶液並推出至廢液的離心管；重覆此步驟 7次。 沖堤樣品：吸取 2.5L編號 3 的沖堤液並推至新的離心管，重複一次。（吸量管仍定在10L，不要改變吸量管的設定，否則會導入空氣至填充的物質中） 抽真空後，並拿出冰冰箱未經 Easytips 處理的離吸管，都加入2L的0.1% TFA。 再加入等體積的介質溶液（matrix），混合好後，取1L的混合液滴至MALDI target plate上。待自然風乾，即可蓋上蓋子。,Matrix for MA
8、LDI-TOF,2,5-Dihydroxybenzoic acid (DHB)Peptides, proteins, lipids, and oligosaccharides3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid) Peptides, proteins, and glycoproteinsa-Cyano-4-hydroxycinnamic acid (CHCA)Peptides, proteins, lipids, and oligonucleotides,Major technique in proteomic researc
9、h: Mass Spectrometry (analysis),Ion source,Mass analyzer,detector,Ion source: substance to ion gasMass analysis: according to mass/charge (m/z)Detection: femtomole attomole (10-15 10-18 mole),Flowchart for protein annotation by Mass spectrometry,Sample Introduction,Mass Spectrum,Peak Assignment,Raw
10、mass data,Postivie Protein ID,參考自鐠得公司,Calculation of the molecular mass,參考自鐠得公司,Nominal Mass,Monoisotopic Mass,Average Mass,C = 12; H = 1; O = 16; N = 14; S = 32,C60H122N20O16S2 = 1442 ; C101H145N34O44 = 2537,C = 12.000; H = 1.00782; O = 15.9949; N = 14.0031; S = 31.9721,C = 12.1115; H = 1.0080; O =
11、 15.9994; N = 14.0067; S = 32.0600,C60H122N20O16S2 = 1442.8788 ; C101H145N34O44 = 2538.0153,C60H122N20O16S2 = 1443.8857 ; C101H145N34O44 = 2539.5,Resolution of Mass,參考自鐠得公司,Resolution : R = m / m,10%,50%,100%,m,m,m1,m2,Using FWHM definition,m / m = 1000,At m/z 1000, m = 1,At m/z 2000, m = 2,1000,100
12、2,m1,2000,m2,2002,m,m,2001,1001,Resolution of Mass,參考自鐠得公司,Ionization source,1.MALDI MSMatrix Assisted Laser Desorption and Ionisation,2.ESI MSElectrospray Ionisation,3.Fast Atom Bombardment (FAB)-MS,1. MALDI MS,參考自鐠得公司,2. ESI MS,參考自鐠得公司,3. Fab MS,參考自鐠得公司,Mass analyzer,參考自鐠得公司,Magnetic Sectors,Quadr
13、upole,Time-Of-Flight (TOF),Q-TOF,TOF-TOF,Triple Quad,IonTrap,FT-ICR,Magnetic Sectors,參考自鐠得公司,Quadrupole,參考自鐠得公司,Triple Quad,CID: Collision Induced Dissociationfor acquiring Molecular weight and Structural information,Time-Of-Flight (TOF),LC Q-TOF,Collision Cell,W Optics,MALDI Q-TOF,MALDI TOF-TOF,Mas
14、s spectrometry-based proteomics,(2003) Aebersold and Mann Nature 422(6928): 198-207,Commonly used Mass Spectrometer in Proteomics,MALDI-TOF,Matrix Assisted Laser Desorption Ionization Time Of Flight,ESI tandem MS (with HPLC, LC tandem MS or LC MS/MS),Electro Spray Ionization Mass Spectrometry,MALDI-
15、TOF,Commercial available MALDI-TOF,Microflex , Bruker,MALDI micro, Micromass,Voyager DE-PRO, ABI,Principal for MALDI-TOF MASS,Two kinds of TOF tubes,Reflectron enhance the resolution,Video for MALDI-Tof,Video for MALDI-Tof (reflectron),Typical result from MALDI-Tof,Maximum allowable Contaminant conc
16、entration (approx.),Peptide mass fingerprinting (PMF),ESI tandem MS or LC/MS/MS,ESI Quadrupole MS,Nano electrospray: 30 min spray time for 1 mL sampleHighly charged molecules are selected by ac modulation of transverse fields,HPLC,Quadrupole mass filter,Typical result from ESI Quadrupole MS,From Eckerskorn in “Bioanalytik”, Lottspeich and Zorbas (Eds),Triple Quadrupole Mass Spectrometer,
