Protein Protocols Protein Protocols

736 Wang and Chait
8. Mass spectrometric matrix solution. 2 mg α-cyano-4-hydroxycinnamic acid, 200 µL 0.1%
TFA in water, 100 µL acetonitrile. Vortex vial thoroughly to suspend the solid into liquid
phase to form turbid mixture and then centrifuge the turbid mixture at 16,000g for 2 min.
The saturated matrix solution will be used in the sample preparation for mass spectrometric
analysis.
3. Methods
3.1. Ladder Generating Reaction by PITC/PIC—Wet Chemistry
This is a manual method to produce ladder peptides based on a manual Edman chemistry
(7) (see Note 4). All of the reactions are carried out in a 0.6-mL polypropylene
microcentrifuge tube. Since PITC is very sensitive to oxidation, some of the operations
are perform under a blanket of dry nitrogen. Operations requiring a nitrogen blanket
will be indicated.
3.1.1. Coupling Reaction
1. Dissolve peptide sample in 20 µL coupling buffer.
2. Add 20 µL coupling reagent (under a blanket of dry nitrogen).
3. Incubate the reaction vial at 50°C for 3 min with a block heater.
3.1.2. Removal of the Coupling/Terminating Reagents
and Byproducts by Two-Phase Liquid Extraction (see Note 5)
1. Remove the reaction vial from the block heater.
2. Wipe the outside of the reaction vial with a prewet paper towel to condense the reaction
liquid.
3. Centrifuge the reaction vial briefly at 16,000g.
4. Add 200 µL of washing solvent A to the reaction vial, vortex gently, and centrifuge at
14,000 rpm for 1 min to clear the phase.
5. Aspirate the upper phase by vacuum suction. Extreme care should be taken to avoid loss
of the lower phase liquid.
6. Repeat steps 4 and 5 one more times with washing solvent A and twice with washing
solvent B. It is advisable to use a fine bore pipet tip for this task.
7. After these four extractions, dry the remaining solution in a Speed-vac centrifuge for
about 5–8 min.
3.1.3. Cleavage Reaction
After the reaction mixture is dried, add 20 µL of anhydrous TFA to the reaction
vial (under a blanket of dry nitrogen). Incubate the reaction vial at 50°C for 2 min
with a block heater.
3.1.4. Removal of Cleavage Reagent
Remove the cleavage reagent, TFA, by drying the reaction vial in a Speed-vac
centrifuge for about 5 min.
3.1.5. Conversion of Free N-Termini to Phenylcarbamyl Derivatives
After the last cycle of coupling-washing-cleavage steps, subject the total peptide
mixture to an additional treatment with PIC to convert any remaining unblocked peptides
to their phenylcarbamyl derivatives.
1. Add 20 µL of conversion buffer and 20 µL of conversion reagent to the reaction vial.
2. Carry out the reaction at 50°C for 5 min.