Peptide-Based Drug Design

Sequence Analysis of Antimicrobial Peptides 43
3. Add 10 �L freshly prepared SPITC solution in a clean vial and aspirate and
dispense this solution three times through the ZipTip TM loaded with the peptide.
Store the ZipTip TM with the SPITC solution on top of the packing material in a
1.5-mL polypropylene vial.
4. Incubate the reaction mixture in the closed vial at 50 ◦ C for 1 h without shaking.
5. Put the ZipTip TM carefully on the pipette again (see Note 19) and dispense the
SPITC solution to the waste.
6. Aspirate 10 �L wash solution into the ZipTip TM and dispense to waste. Repeat
this procedure seven times without getting air into the packed material.
7. Add 2 �L elution solution to a clean 0.6-mL polypropylene vial and aspirate and
dispense it five times through the ZipTip TM without getting air into the stationary
phase.
8. Use the sample for MALDI-MS with CHCA and record the spectra in positive
and negative ion mode.
9. The increment mass of the N-terminal modification is 214.97 u.
10. For sequence analysis record the MS/MS spectra (Fig. 3) for all modified
peptides in positive ion mode (see Note 20).
3.8.3. N-Terminal Derivatization with 2-Sulfobenzoic Acid
Cyclic Anhydride
1. Dissolve the dried peptide sample in 2 �L reaction buffer.
2. Add 2 �L freshly prepared SACA reagent (see Note 21) and mix.
3. Incubate at 24 ◦ C for 5 min in the thermomixer (800 rpm).
4. Prepare the sample for MALDI-MS with CHCA and record the spectra in positive
and negative ion mode (see Note 20).
5. The increment mass of the N-terminal modification is 184.17 u.
6. For sequence analysis record the MS/MS spectra for all modified peptides in
positive ion mode (see Note 20).
4. Notes
1. If a reversed-phase HPLC is coupled on-line to a mass spectrometer equipped
with an electrospray ionization source, it is important to use acetonitrile with “MS
grade” purity instead of “HPLC grade” or “UV grade” to reduce the background
noise in the m/z range above 200. Otherwise several strong signals are obtained
that may disturb data processing. The eluents should be replaced weekly. We use
only “MS grade” acetonitrile to prepare buffers and reagents for MS analysis.
2. The addition of TFA or other volatile acids to the matrix improves the peptide
ionization, increasing the signal intensities. Alternatively, the acid can be added
to the peptide sample. If the sample already contains a high acid content it is
better to add pure matrix without TFA to prevent cluster formation increasing the
background noise.