Peptide-Based Drug Design

22 Bulet
interest. As no instrument offers all capabilities simultaneously, complementary
mass spectrometers (ESI and MALDI) are often required for peptidomics and
proteomics studies. See Note 14 for additional comments on MS.
3.7.1. MALDI-TOF
For MALDI-TOF MS several sample preparations are available with different
matrices (26). The choice of the matrix and of the sample preparation should
be adapted to the molecular mass of the compounds and to the complexity of
the samples to analyze. �-Cyano-4-hydroxycinnamic acid (4HCCA) is preferred
for peptides between 1 and 15 kDa, and the sandwich sample preparation can
be universally used for molecular mass determination of pure peptides and
enzymatic fragments or complex mixtures (e.g., crude hemolymph, enzymatic
digests). The procedures reported below are the ones used for the discovery of
the Drosophila immune-induced peptides (19,27,30).
1. 0.5 �L of a saturated solution of 4HCCA in acetone is placed on the MALDI
sample plate.
2. When this is dried, deposit 0.5 �L of 1% TFA on the crystallized matrix bed.
3. For molecular mass determination, dissolve/dilute the purified peptide in acidified
water (0.5% TFA) and deposit 0.5 �L of the acidified peptide solution.
4. Immediately add a droplet of 0.2 �L of a saturated solution of matrix 4HCCA in
50% acetonitrile in acidified water.
5. Dry the target under gentle vacuum and wash (also used for desalting if crude
hemolymph or digestion mixture) the sample preparation twice with 1 �L of1%
TFA. Remove the washing solution after a few seconds using forced air, and dry
the sample under vacuum.
6. Insert the sample probe in the MALDI-TOF mass spectrometer. Calibration is
performed in external mode with peptides covering the mass range of 500 Da to 5
kDa (see Note 15).
3.7.2. ESI-MS & -MS n
1. Dissolve the fraction aliquot to analyze in a solution of water/acetonitrile/formic
acid (composition 49.9/49.9/0.2, v/v/v) (see Note 16).
2. Inject, using a Hamilton syringe (total volume of the syringe 5 �L), a fivefold
dilution first for evaluating the intensity of the mass signals. If necessary, inject
the rest of the sample.
3. For nano-ESI-MS & MS n , the sample is loaded either in a previously rinsed
gold/palladium-coated nanospray capillary (Promega) or in a PicoTip TM emitter
(New Objective) and analyzed in a nanospray source.
4. Calibrate the mass analyzer with the multicharged ions of at least three to five
standard peptides or following Note 15.