2.6 Solid-Phase
Peptide Synthesis
1. 2-(tritylmercapto)acetyl-L-leucinyl-PAM (Trt-S-Ac-L-Leu-PAM)
resin for Boc-based synthesis of peptide thioesters.
2. 2-clorotrityl chloride (2-CTC) resin on polystyrene for Fmocbased chemical synthesis of peptide hydrazides.
3. Preloaded Wang resin for Fmoc-based synthesis.
4. Fmoc-PAL-PEG-PS™ for Fmoc-based chemical synthesis of
long peptides.
5. Phenylacetamidomethyl (PAM) resin for Boc-based synthesis.
6. Fmoc/Boc-protected canonical amino acids.
7. Fmoc-Ser(PO(OBzl)OH)-OH, Fmoc-Thr(PO(OBzl)OH)OH, Fmoc-Tyr(PO(OBzl)OH)-OH for phosphopeptides.
8. α-hydroxy amino acids (see Note 2) for depsipeptides.
9. 2-(1-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) prepared as a 0.5 M stock solution
in DMF in a light-protected bottle.
10. Diisopropylamine (DIEA).
11. 20% (v/v) piperidine in DMF.
12. Fmoc-cleavage mix: 82.5% (v/v) TFA, 5% (v/v) phenol, 5%
(v/v) H 2 O, 5% (v/v) thioanisole, 2.5% (v/v) 1,2-ethanedithiol
(EDT). Prepare fresh.
13. Peptide dissolution buffer: 20% MeCN, 0.1% TFA.
14. Ninhydrin test/Kaiser test kit.
15. Disposable Reaction Tube (10 mL) (SiliCycle Inc.).
2.7 Peptide
Purification
1. High-pressure liquid chromatography (HPLC) buffer A: 5%
(v/v) acetonitrile (MeCN), 0.1% (v/v) TFA in ultrapure water,
filtered with 0.2 μm filters.
2. HPLC Buffer B: 95% MeCN, 0.1% TFA in ultrapure water,
filtered with 0.2 μm filters.
2.8 Expressed
Protein Ligation
1. NaNO 2 stock solution: 0.2 M in ultrapure water. Prepare fresh.
2. Dithiothreitol (DTT) stock solution: 1 M in ultrapure water.
Can be stored at À20
C.
3. 300 mM Tris(2-carboxyethyl)phosphine hydrochloride
(TCEP) stock solution in ultrapure water.
4. Ligation buffer 1: 0.2 M sodium phosphate pH 3.0, 6 M
guanidinium hydrochloride (Gu·HCl).
5. Ligation buffer 2: 0.2 M 4-mercaptophenylacetic acid (MPAA)
in 6 M Gu·HCl, 0.2 M sodium phosphate pH 7.0. Prepare
fresh.
200
Christin Kossmann et al.
Peptide Synthesis
1. 2-(tritylmercapto)acetyl-L-leucinyl-PAM (Trt-S-Ac-L-Leu-PAM)
resin for Boc-based synthesis of peptide thioesters.
2. 2-clorotrityl chloride (2-CTC) resin on polystyrene for Fmocbased chemical synthesis of peptide hydrazides.
3. Preloaded Wang resin for Fmoc-based synthesis.
4. Fmoc-PAL-PEG-PS™ for Fmoc-based chemical synthesis of
long peptides.
5. Phenylacetamidomethyl (PAM) resin for Boc-based synthesis.
6. Fmoc/Boc-protected canonical amino acids.
7. Fmoc-Ser(PO(OBzl)OH)-OH, Fmoc-Thr(PO(OBzl)OH)OH, Fmoc-Tyr(PO(OBzl)OH)-OH for phosphopeptides.
8. α-hydroxy amino acids (see Note 2) for depsipeptides.
9. 2-(1-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) prepared as a 0.5 M stock solution
in DMF in a light-protected bottle.
10. Diisopropylamine (DIEA).
11. 20% (v/v) piperidine in DMF.
12. Fmoc-cleavage mix: 82.5% (v/v) TFA, 5% (v/v) phenol, 5%
(v/v) H 2 O, 5% (v/v) thioanisole, 2.5% (v/v) 1,2-ethanedithiol
(EDT). Prepare fresh.
13. Peptide dissolution buffer: 20% MeCN, 0.1% TFA.
14. Ninhydrin test/Kaiser test kit.
15. Disposable Reaction Tube (10 mL) (SiliCycle Inc.).
2.7 Peptide
Purification
1. High-pressure liquid chromatography (HPLC) buffer A: 5%
(v/v) acetonitrile (MeCN), 0.1% (v/v) TFA in ultrapure water,
filtered with 0.2 μm filters.
2. HPLC Buffer B: 95% MeCN, 0.1% TFA in ultrapure water,
filtered with 0.2 μm filters.
2.8 Expressed
Protein Ligation
1. NaNO 2 stock solution: 0.2 M in ultrapure water. Prepare fresh.
2. Dithiothreitol (DTT) stock solution: 1 M in ultrapure water.
Can be stored at À20
C.
3. 300 mM Tris(2-carboxyethyl)phosphine hydrochloride
(TCEP) stock solution in ultrapure water.
4. Ligation buffer 1: 0.2 M sodium phosphate pH 3.0, 6 M
guanidinium hydrochloride (Gu·HCl).
5. Ligation buffer 2: 0.2 M 4-mercaptophenylacetic acid (MPAA)
in 6 M Gu·HCl, 0.2 M sodium phosphate pH 7.0. Prepare
fresh.
200
Christin Kossmann et al.