1. 蛋白質體學
Proteomics 2011
Solid-Phase Peptide Synthesis (SPPS) and Applications of Synthetic Peptides
陳威戎
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。

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
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

6. Solid Support - Resin
Resin for SPPS: polystyrene bead with 1% divinyl-benzene, a cross-linking agent.
Dry resin beads: microns, or 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
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

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) 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

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
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

24. HPLC- Purity Analysis

25. Amino Acid Composition Analysis
Column : Pico Tag for amino acid composition analysis (Waters)
Eluent A : 0.1 M NH4OAc, 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

26. Amino Acid Composition Analysis-Standard

27. Amino Acid Composition Analysis-Sample

28. 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
Leu
183749
935.00
Phe
150162
560.65
Lys
229405
757.62
68605 91
1.3

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: 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
Peptide Array
Stimulus-Responsive Peptides

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

39. Antimicrobial Peptides (AMPs) ~ Host-Defense Peptides (HDPs)

49. Peptide Vaccine

50. Peptide Vaccine

51. Peptide Vaccine

57. Peptide Array

64. Stimulus-Responsive Peptides

65. Applications of Stimulus-Responsive Peptides
Chockalingam, K. et al.
Protein Engineering, Design and Selection : ; doi: /protein/gzm008

68. Stimulus-Responsive Peptides

72. Table 1
Summary of drug-delivery vehicles and stimulus-responsive drug carriers that have been developed to increase targeted accumulation of a drug, enhance its distribution, and control its intracellular or subcellular localization, with the ultimate goal of increasing the efficacy of anticancer therapeutics.
Drug carrier
Stimulus response
Drug
Application
Abraxane® (albumin–drug conjugate) –
Paclitaxel
Clinically approved for the treatment of breast cancer
NK105 (drug-loaded PEG–poly(aspartic acid) micelle)
In clinical trials for the treatment of colon and stomach cancers
Doxil® (drug-loaded PEGylated liposome)
Doxorubicin
Clinically approved for the treatment of recurrent ovarian cancer
ELP unimer
Temperature-triggered aggregation
Increased accumulation in hyperthermia treated tumors in mice
Dextran–peptide–drug conjugate
MMP cleavage of peptide linker for release of free drug
Methotrexate
Improved inhibition of tumor growth in subcutaneous murine models of human fibrosarcoma and glioblastoma
Poly(histidine)-β-PEG, poly(l-lactic acid)-β-PEG mixed micelle
pH-triggered disassembly for release of free drug
Increased accumulation and improved efficacy in subcutaneous breast cancer tumors in mice
Pluronic® micelle
Ultrasound-induced disassembly for release of free drug
Improved regression with ultrasound treatment in subcutaneous colon cancer tumors in mice in comparison to treatment with drug-loaded micelles alone
Oligoarginine–peptide–oligoglutamate conjugate
MMP-2 cleavage of peptide linker for CPP activation
Increased accumulation compared with uncleavable controls in a variety of subcutaneously implanted tumor types in mice
PEG–poly(l-histidine), poly(l-lactic acid)–PEG–poly(l-histidine)–TAT mixed micelle
pH-triggered display of TAT for CPP activation
Selective cellular uptake in slightly acidic conditions of pH 7.0 improved cytotoxicity in drug-resistant breast cancer cells
RGD-functionalized ELP diblock
Temperature-triggered micelle assembly for polyvalent ligand display
Increased accumulation with hyperthermia-triggered micelle assembly in leukemia cells overexpressing αvβ3 integrin
Thiolated heparin nanogel
Destabilization of disulfide bonds in reducing intracellular environment for free drug release
Heparin
Increased cytotoxicity by induction of apoptosis in melanoma cells as compared with free drug
ELP–drug conjugate micelle
pH-triggered drug release in acidic endosomal compartment
Enhanced accumulation, increased MTD and improved regression of colon carcinoma tumors in mice
Poly(l-histidine)-based micelle
pH-triggered protonation of histidine for endosomal disruption
Improved cytotoxicity of intracellularly released drug in doxorubicin-resistant ovarian carcinoma cells
Amidized poly(l-lysine)–drug conjugate
pH-triggered charge reversal for nuclear targeting
Camptothecin
Nuclear localization of drug-enhanced cytotoxicity, compared with free drug, in adenocarcinoma cells
TPP-modified liposome
Ceramide
Improved inhibition of tumor growth with mitochondrial targeting in a subcutaneous mouse model of mammary carcinoma tumors