蛋白質體學 Proteomics 2018 Solid-Phase Peptide Synthesis (SPPS) and Applications of Synthetic Peptides 陳威戎 2018.12.03 純化酵素是一件非常基本的工作，很多重要的研究，都脫不開酵素的純化工作。而大多數酵素的純化，基本上也脫不開一些最基本的原則。 首先，建立一個完善的酵素實驗室是很必要的；我們把許多實驗室內的運作細節一一交代，期望同學能認知這些經驗，確實接收並養成習慣，且期望應用到將來每個人的研究工作上。 最先遇到，但是最容易被忽視的步驟，就是材料處理及總蛋白質的抽取。將提醒你小心選擇採料的種類、時期、部位等，並選擇一個良好的粗抽取方式，以便有一個良好的開始。
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
Strategies for SPPS Chain Assembly Boc (t-butyloxycarbonyl) Fmoc (9-fluorenylmethoxycarbonyl)
Protecting Group Strategies in SPPS
Comparison of Boc and Fmoc SPPS Requires special equipment Yes No Cost of reagents Lower Higher Solubility of peptides Purity of hydrophobic peptides High May be lower Problems with aggregation Less frequently More frequently Synthesis time ~20 min/amino acid ~20-60 min/amino acid Final deprotection HF TFA Safety Potentially dangerous Relatively safe
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.
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)
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)
General Protocols - Fmoc chemistry Loading Deprotection wash Activation Coupling wash Repeat ~ Cleavage from resin
General Protocols- Fmoc chemistry
Loading and Capping DCC (N,N’-dicyclohexylcarbodiimide) DMAP (4-Dimethylaminopyridine) Acetic (benzoic) anhydride
Deprotection - Piperidine Conductivity monitoring
Coupling Efficiency Vs. Peptide Length 0.995 0.99 0.98 0.97 0.96 5 0.95 0.92 0.89 0.85 10 0.91 0.83 0.76 0.69 15 0.93 0.87 0.75 0.65 0.56 20 0.68 0.46 25 0.79 0.62 0.48 0.38 30 0.86 0.41 0.31 35 0.84 0.71 0.50 0.36 0.25 40 0.82 0.67 0.45 0.30 0.20 45 0.80 0.63 0.26 0.17 50 0.78 0.60 0.37 0.22 0.14 55 0.58 0.34 0.19 0.11 60 0.74 0.55 0.09 65 0.73 0.53 0.27 0.07 70 0.12 0.06
Activation – HBTU/HOBt HBTU: 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium HOBt: 1-hydroxybenzotriazole HBTU activation ~ FastMoc chemistry !
Coupling Objectives: maximize solvation and minimize hydrogen bonding DMF (dimethylformamide) ; NMP (N-methylpyrrolidone) DIEA (diisopropylethylamine)
Cleavage from Resin and Removal of Side-Chain Protecting groups
TFA Cleavage – 95% TFA + Scavengers
Scavengers
Purification Filtration and DCM wash Concentration by Rotavapor Ether extraction Lyophilization Purification by HPLC
Additional Chemical Modification Disulfide bond formation Phosphorylation Biotinylation Farnesylation Glycosylation C- and N-terminal modification Chromophore and fluorophore labelling
How to Choose Peptide Solvents Peptides with a net positive charge: (1) H2O alone (2) gently shake / warm up to 30oC (3) 10% HOAc Peptides with a net negative charge: (1) H2O or HOAc (2) NH4HCO3 Peptides with a net zero charge: (1) H2O, HOAc, warming and shaking (2) 6M guanidine-HCl, TFA, HCOOH (3) MeOH, isopropanol, acetonitrile
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
HPLC- Purity Analysis Column : JUPITER 5u C18, 250 x 4.60 mm , 300 Å (phenomenex) Eluent A : 0.1% TFA Eluent B : 0.08% TFA in 80% CH3CN Gradient : Time (min) Flow rate (ml/min) Eluent A (%) Eluent B Initial 1.00 100 30.00 40.00 40.01 0.00 (5) Sample preparation : appropriate amount in d.d. H2O (6) Loading : 1 mL
HPLC- Purity Analysis
Amino Acid Composition Analysis Column : Pico Tag for amino acid composition analysis (Waters) Eluent A : 0.1 M NH4OAc, 0.03 M NH4(SO4)2, 0.04% AcOH Eluent B : 0.1 M NH4OAc, 50% CH3CN Gradient : Time (min) Flow rate (ml/min) Eluent A (%) Eluent B Initial 1.00 100 10.00 95 5 35.00 45 55 42.00 0.00 42.01 (5) Sample preparation : appropriate amount in 2 mM NaOH (6) Loading : 20 mL
Amino Acid Composition Analysis-Standard
Amino Acid Composition Analysis-Sample
Amino Acid Composition Analysis a.a. standard area pmol area/pmol sample area pmole 理論值 實驗值 Asx 146761 250 587.04 116669 199 3 2.9 Glx 131146 524.58 105742 202 Ser 141532 566.13 29319 52 1 0.7 Gly 156800 627.20 142010 226 3.3 His 145333 581.33 40358 69 1.0 Thr 145038 580.15 0.0 Ala 159585 638.34 Arg 152570 610.28 Pro 153833 615.33 Tyr 157653 630.61 82851 131 2 1.9 Val 160931 643.72 43161 67 Met 155710 582.84 82829 142 2.0 Ile 177989 1471.96 Leu 183749 935.00 Phe 150162 560.65 Lys 229405 757.62 68605 91 1.3
ABI 433A Peptide Synthesizer
ABI 433A – Front View
ABI 433A – Rear View
ABI 433A – Flow Schematics
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
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
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
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