2. Receptor capture: PBS pH 6.5: Acidify 500 mL of PBS
(pH 7.4) by adding 105 μL of 85% (vol/vol) phosphoric acid.
3. Sodium (meta) periodate 1.5 mM: Dissolve 16 mg of sodium
(meta) periodate in 50 mL of PBS pH 6.5 and vortex the
solution until it is fully dissolved (always prepare fresh, protect
from light).
4. 5-Methoxyanthranilic acid 5 mM: Dissolve 84 mg of
5-methoxyanthranilic acid in 100 mL PBS pH 7.4; stir with
heating at 40
C until it is fully dissolved and adjust pH to 7.4
with NaOH (protect from light).
5. Lysis buffer: Dissolve 4.8 g urea to 10 mL deionized water, stir
until it is fully dissolved. Add 0.1% (vol/vol) Rapigest SF and
one Protease Inhibitors cOmplete tablet, stir until it is fully
dissolved (protect from light).
6. Protein enrichment: CuSO 4 100 mM: Dissolve 25 mg of
CuSO4 to 1 mL deionized water.
7. THPTA 100 mM: Dissolve 44 mg of THTPA to 1 mL deionized water (store aliquots in À20
C).
8. Sodium ascorbate 1 M: Dissolve 200 mg of sodium ascorbate
in 1 mL of deionized water (prepare fresh).
9. Click Chemistry Master Mix (2Â): In 835 μL deionized water,
add 125 μL THPTA, 20 μL CuSO4, and vortex; then add
20 μL sodium ascorbate and vortex again.
10. Tris-HCl 1 M: Dissolve 121.1 g of Tris base in 800 mL of
deionized water; adjust the pH to 8.0 with HCl.
11. NaCl 5 M: Dissolve 292gm of NaCl in 1 L of deionized water.
12. EDTA 100 mM: Dissolve 1.86 g of EDTA to 50 mL of
deionized water and adjust pH to 7.4 with NaOH.
13. SDS wash buffer (1% SDS, 100 mM Tris, 250 mM NaCl,
5 mM EDTA, pH 8): Mix 10 mL 1 M Tris–HCL, 5 mL
NaCl, 5 mL 100 mM EDTA, and 1gm of SDS in a final volume
of 100 mL.
14. Urea wash buffer (8 M urea in 100 mM Tris pH 8): Mix 48 g
urea and 10 mL 1 M Tris to a final volume of 100 mL.
15. Isopropanol 80%: Mix 80 mL of isopropanol and 20 mL deionized water.
16. NaHCO 3 100 mM: Dissolve 84 mg sodium bicarbonate in
100 mL deionized water, and adjust the pH to 11.0
with NaOH.
17. Acetonitrile 20%: Mix 20 mL of acetonitrile and 80 mL of
deionized water.
18. Ammonium bicarbonate 50 mM: Dissolve 39 mg of ammonium bicarbonate in 10 mL of deionized water.
Validating Cardiac Targeting Peptide
103
(pH 7.4) by adding 105 μL of 85% (vol/vol) phosphoric acid.
3. Sodium (meta) periodate 1.5 mM: Dissolve 16 mg of sodium
(meta) periodate in 50 mL of PBS pH 6.5 and vortex the
solution until it is fully dissolved (always prepare fresh, protect
from light).
4. 5-Methoxyanthranilic acid 5 mM: Dissolve 84 mg of
5-methoxyanthranilic acid in 100 mL PBS pH 7.4; stir with
heating at 40
C until it is fully dissolved and adjust pH to 7.4
with NaOH (protect from light).
5. Lysis buffer: Dissolve 4.8 g urea to 10 mL deionized water, stir
until it is fully dissolved. Add 0.1% (vol/vol) Rapigest SF and
one Protease Inhibitors cOmplete tablet, stir until it is fully
dissolved (protect from light).
6. Protein enrichment: CuSO 4 100 mM: Dissolve 25 mg of
CuSO4 to 1 mL deionized water.
7. THPTA 100 mM: Dissolve 44 mg of THTPA to 1 mL deionized water (store aliquots in À20
C).
8. Sodium ascorbate 1 M: Dissolve 200 mg of sodium ascorbate
in 1 mL of deionized water (prepare fresh).
9. Click Chemistry Master Mix (2Â): In 835 μL deionized water,
add 125 μL THPTA, 20 μL CuSO4, and vortex; then add
20 μL sodium ascorbate and vortex again.
10. Tris-HCl 1 M: Dissolve 121.1 g of Tris base in 800 mL of
deionized water; adjust the pH to 8.0 with HCl.
11. NaCl 5 M: Dissolve 292gm of NaCl in 1 L of deionized water.
12. EDTA 100 mM: Dissolve 1.86 g of EDTA to 50 mL of
deionized water and adjust pH to 7.4 with NaOH.
13. SDS wash buffer (1% SDS, 100 mM Tris, 250 mM NaCl,
5 mM EDTA, pH 8): Mix 10 mL 1 M Tris–HCL, 5 mL
NaCl, 5 mL 100 mM EDTA, and 1gm of SDS in a final volume
of 100 mL.
14. Urea wash buffer (8 M urea in 100 mM Tris pH 8): Mix 48 g
urea and 10 mL 1 M Tris to a final volume of 100 mL.
15. Isopropanol 80%: Mix 80 mL of isopropanol and 20 mL deionized water.
16. NaHCO 3 100 mM: Dissolve 84 mg sodium bicarbonate in
100 mL deionized water, and adjust the pH to 11.0
with NaOH.
17. Acetonitrile 20%: Mix 20 mL of acetonitrile and 80 mL of
deionized water.
18. Ammonium bicarbonate 50 mM: Dissolve 39 mg of ammonium bicarbonate in 10 mL of deionized water.
Validating Cardiac Targeting Peptide
103