Detection of Specific Zinc Finger Peptide Complexes with Matrix-Assisted Laser Desorption
141
The complexation behavior in solution was probed by circular dichroism (CD)
spectroscopy because the peptides secondary structure elements show characteristic CD spectra (Fasman 1996).
Fig. 9.2 depicts the MALDI mass spectra of p55F1 with ATT as a matrix. A signal of the protonated molecular ion is observed (Fig. 9.2A). Upon addition of 10
molar equivalents of Zn 2 +, a signal corresponding to the peptide adduct with
Zn 2 + appears (Fig. 9.2B). The reasons why a large excess of Zn 2 + is needed to see
a significant zinc complex signal in MALDI, whereas CD spectra already show a
complete formation of the Zn 2 + -peptide complex in solution in a 1: 1 molar ratio,
are explained in detail in the paper of Lehmann et al. 1998.
In the following, the ratio of the peak integrals of the Zn 2 + -adduct and the protonated peptide will be called relative Zn 2 +-adduct intensity. The Zn-p55F1 signal
may either represent a specific 1:1 complex or a nonspecific adduct. To distinguish between both possibilities, the peptide gramicidin S, which does not form
specific adducts with Zn 2 +, was added to the sample (Fig. 9.2C). The relative
Zn 2 + -adduct intensity of p55F1 is the same as without addition of gramicidin S.
The Zn 2 + -adduct of gramicidin S is negligible. Therefore, this experiment indicates a specific complex of Zn 2 + with p55Fl.
The association reaction of the Zn 2 +-p55F1 complex in solution can be written
as follows:
p55F1 + Zn 2 + :;:= [(p55Fl) + Znf+
(1)
The formation constant is 1010 M- l • The affinities of p55F1 for other metal ions
are assumed to be lower, but are not known. Therefore, they were qualitatively
Fig. 9.2. MALDI mass spectra with ATT
matrix of (A) peptide p55FI alone, (B) peptide and Zn 2 + in molar ratio I: 1 0, and (C)
peptide and Zn2+ in molar ratio I: 1 0 with
addition of gramicidin S. Solvent: water, pH
5. The spectra are normalized to the [P+Hj+
signal (adapted with permission from Lehmann et al. 1999)
.
I peptide, Zn 2 + and gramicidin S
+
~
8
peptide and Zn 2 +
~
I ------------~----~
I _____ p_ ep _t_i d_e ________ ~
I
I
I
I
I
I
1200 1400 1600 1800 2000 2200 2400
m/z
141
The complexation behavior in solution was probed by circular dichroism (CD)
spectroscopy because the peptides secondary structure elements show characteristic CD spectra (Fasman 1996).
Fig. 9.2 depicts the MALDI mass spectra of p55F1 with ATT as a matrix. A signal of the protonated molecular ion is observed (Fig. 9.2A). Upon addition of 10
molar equivalents of Zn 2 +, a signal corresponding to the peptide adduct with
Zn 2 + appears (Fig. 9.2B). The reasons why a large excess of Zn 2 + is needed to see
a significant zinc complex signal in MALDI, whereas CD spectra already show a
complete formation of the Zn 2 + -peptide complex in solution in a 1: 1 molar ratio,
are explained in detail in the paper of Lehmann et al. 1998.
In the following, the ratio of the peak integrals of the Zn 2 + -adduct and the protonated peptide will be called relative Zn 2 +-adduct intensity. The Zn-p55F1 signal
may either represent a specific 1:1 complex or a nonspecific adduct. To distinguish between both possibilities, the peptide gramicidin S, which does not form
specific adducts with Zn 2 +, was added to the sample (Fig. 9.2C). The relative
Zn 2 + -adduct intensity of p55F1 is the same as without addition of gramicidin S.
The Zn 2 + -adduct of gramicidin S is negligible. Therefore, this experiment indicates a specific complex of Zn 2 + with p55Fl.
The association reaction of the Zn 2 +-p55F1 complex in solution can be written
as follows:
p55F1 + Zn 2 + :;:= [(p55Fl) + Znf+
(1)
The formation constant is 1010 M- l • The affinities of p55F1 for other metal ions
are assumed to be lower, but are not known. Therefore, they were qualitatively
Fig. 9.2. MALDI mass spectra with ATT
matrix of (A) peptide p55FI alone, (B) peptide and Zn 2 + in molar ratio I: 1 0, and (C)
peptide and Zn2+ in molar ratio I: 1 0 with
addition of gramicidin S. Solvent: water, pH
5. The spectra are normalized to the [P+Hj+
signal (adapted with permission from Lehmann et al. 1999)
.
I peptide, Zn 2 + and gramicidin S
+
~
8
peptide and Zn 2 +
~
I ------------~----~
I _____ p_ ep _t_i d_e ________ ~
I
I
I
I
I
I
1200 1400 1600 1800 2000 2200 2400
m/z