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Proteomics 2012 蛋白質體學 Solid-Phase Peptide Synthesis (SPPS) and Applications of Synthetic Peptides 陳威戎.

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Presentation on theme: "Proteomics 2012 蛋白質體學 Solid-Phase Peptide Synthesis (SPPS) and Applications of Synthetic Peptides 陳威戎."— Presentation transcript:

1 Proteomics 2012 蛋白質體學 Solid-Phase Peptide Synthesis (SPPS) and Applications of Synthetic Peptides 陳威戎

2 Solid-Phase Peptide Synthesis (SPPS) Chain assembly Cleavage from resin and removal of side-chain protecting groups Purification Additional chemical modification Characterization ~ first introduced by Bruce Merrifield in 1963

3 Strategies for SPPS Chain Assembly Boc (t-butyloxycarbonyl) Fmoc (9-fluorenylmethoxycarbonyl)

4 Protecting Group Strategies in SPPS

5 Comparison of Boc and Fmoc SPPS BocFmoc Requires special equipment YesNo Cost of reagentsLowerHigher Solubility of peptidesHigherLower Purity of hydrophobic peptides HighMay be lower Problems with aggregation Less frequentlyMore frequently Synthesis time~20 min/amino acid ~20-60 min/amino acid Final deprotectionHFTFA Safety Potentially dangerous Relatively safe

6 Solid Support - Resin Resin for SPPS: polystyrene bead with 1% divinyl-benzene, a cross-linking agent. Dry resin beads: 40-100 microns, 100-200 or 200-400 mesh When in contact with solvents, the beads swell to approximately 10 times their dry volume. Macroscopically, the resin appears as an insoluble solid support. However, on the molecular level the resin is “in solution” or fully solvated. This solvation enhances coupling of the peptide resin with the protected amino acids.

7 Fmoc Resins HMP resin (4-hydroxymethyl-phenoxymethyl-copolystyrene-1% divinylbenzene resin), also known as Wang resin produces a carboxylic acid terminal peptide Amide resin – produces an amide terminal peptide MAPS resin (multiple antigenic peptides resin)

8 Protected Fmoc Amino Acid Derivatives Asp(OtBu) ; Glu(OtBu) ; Asn(Trt) ; Gln(Trt) Arg(Pmc) ; His(Trt) ; Lys(Boc) Ser(tBu) ; Thr(tBu) ; Tyr(tBu) ; Cys(Trt)

9 General Protocols - Fmoc chemistry  Loading  Deprotection wash Activation  Coupling wash Repeat  ~  Cleavage from resin

10 General Protocols- Fmoc chemistry

11 Loading and Capping DCC (N,N’-dicyclohexylcarbodiimide) DMAP (4-Dimethylaminopyridine) Acetic (benzoic) anhydride

12 Deprotection - Piperidine Conductivity monitoring

13 Coupling Efficiency Vs. Peptide Length Peptide Length Coupling Efficiency 00.9950.990.980.970.96 50.980.950.920.890.85 100.960.910.830.760.69 150.930.870.750.650.56 200.910.830.680.560.46 250.890.790.620.480.38 300.860.750.560.410.31 350.840.710.500.360.25 400.820.670.450.300.20 450.800.630.410.260.17 500.780.600.370.220.14 550.760.580.340.190.11 600.740.550.300.170.09 650.730.530.270.140.07 700.710.500.250.120.06

14 Activation – HBTU/HOBt HBTU: 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium HOBt: 1-hydroxybenzotriazole HBTU activation ~ FastMoc chemistry !

15 Coupling Objectives: maximize solvation and minimize hydrogen bonding DMF (dimethylformamide) ; NMP (N-methylpyrrolidone) DIEA (diisopropylethylamine)

16 Cleavage from Resin and Removal of Side-Chain Protecting groups

17 TFA Cleavage – 95% TFA + Scavengers

18 Scavengers

19 Purification Filtration and DCM wash Concentration by Rotavapor Ether extraction Lyophilization Purification by HPLC

20 Additional Chemical Modification Disulfide bond formation Phosphorylation Biotinylation Farnesylation Glycosylation C- and N-terminal modification Chromophore and fluorophore labelling

21 How to Choose Peptide Solvents Peptides with a net positive charge: (1) H 2 O alone (2) gently shake / warm up to 30 o C (3) 10% HOAc Peptides with a net negative charge: (1) H 2 O or HOAc (2) NH 4 HCO 3 Peptides with a net zero charge: (1) H 2 O, HOAc, warming and shaking (2) 6M guanidine-HCl, TFA, HCOOH (3) MeOH, isopropanol, acetonitrile

22 Characterization Purity analysis by HPLC Amino acid composition analysis by precolumn PITC derivatization on a PicoTag HPLC system Determination of peptide molecular weight by mass spectrometry

23 HPLC- Purity Analysis (1) Column : JUPITER 5u C18, 250 x 4.60 mm, 300 Å (phenomenex) (2) Eluent A : 0.1% TFA (3) Eluent B : 0.08% TFA in 80% CH 3 CN (4) Gradient : Time (min) Flow rate (ml/min) Eluent A (%) Eluent B (%) Initial1.001000 30.001.000100 40.001.000100 40.010.000100 (5) Sample preparation : appropriate amount in d.d. H 2 O (6) Loading : 1 mL

24 HPLC- Purity Analysis

25 Amino Acid Composition Analysis (1) Column : Pico Tag for amino acid composition analysis (Waters) (2) Eluent A : 0.1 M NH 4 OAc, 0.03 M NH 4 (SO 4 ) 2, 0.04% AcOH (3) Eluent B : 0.1 M NH 4 OAc, 50% CH 3 CN (4) Gradient : Time (min) Flow rate (ml/min) Eluent A (%) Eluent B (%) Initial1.001000 10.001.00955 35.001.004555 42.000.000100 42.010.000100 (5) Sample preparation : appropriate amount in 2 mM NaOH (6) Loading : 20  L

26 Amino Acid Composition Analysis-Standard

27 Amino Acid Composition Analysis-Sample

28 Amino Acid Composition Analysis a.a.standard areapmolarea/pmolsample areapmole 理論值實驗值 Asx146761250587.0411666919932.9 Glx131146250524.5810574220232.9 Ser141532250566.13293195210.7 Gly156800250627.2014201022633.3 His145333250581.33403586911.0 Thr145038250580.150000.0 Ala159585250638.340000.0 Arg152570250610.280000.0 Pro153833250615.330000.0 Tyr157653250630.618285113121.9 Val160931250643.72431616711.0 Met155710250582.848282914222.0 Ile1779892501471.960000.0 Leu183749250935.000000.0 Phe150162250560.650000.0 Lys229405250757.62686059111.3

29 ABI 433A Peptide Synthesizer

30 ABI 433A – Front View

31 ABI 433A – Rear View

32 ABI 433A – Flow Schematics

33 PS3 Peptide Synthesizer - PTI PS3- a lot cheaper and easier to use! - Simple and fast cycle time under 40 mins/coupling - Variety of coupling techniques - Zero-dead-volume fluid valve system - Self diagnostic program - Higher productivity up to 45 couplings automatically 3 different peptides sequentially

34 Symphony Peptide Synthesizer - PTI Symphony/Multiplex 12-channel solid- phase synthesizer - Fast multiplex operation operate 12-channel simultaneously - Patented multiplexing matrix valve - Lower coupling reagent cost - Variable scales: 0.005-0.35 mmol - Automated cleavage - Easily customized protocols - Extreme versatility

35 Microwave Peptide Synthesizer - CEM Odyssey System on a Discover platform World’s first microwave peptide synthesizer wins 2004 R&D 100 Award! -Significantly increased reaction rates cycle time less than 10 mins - Better product purity and yield - Overcoming chain aggregation - Automated cleavage within 15 mins - Lower cost: cheaper reagents - Useful on multiple programmable scale - Greater flexibility

36 PepSy Peptide Synthesizer - Zinsser Parallel synthesis of peptide libraries in 96-well plate format. - 9 independent 96-well reactor stations - 864 peptides in 30 h, 10 mer, ~ 1 mg each - Dispensing pen for each a.a. - no washes or flushes needed - speeds up synthesis - no cross contamination - Bar code check for every step - Software-assisted library design

37 Applications of Synthetic Peptides Antimicrobial Peptides (AMPs) ~ Host-Defense Peptides (HDPs) Peptide Vaccine Stimulus-Responsive Peptides

38 Antimicrobial Peptides (AMPs) ~ Host-Defense Peptides (HDPs)

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49 Peptide Vaccine

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53 Stimulus-Responsive Peptides

54 Applications of Stimulus-Responsive Peptides Chockalingam, K. et al. Protein Engineering, Design and Selection 2007 20:155-161; doi:10.1093/protein/gzm008

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