Detection of Specific Zinc Finger Peptide Complexes with Matrix-Assisted Laser Desorption
143
ments show that the peptide complex is completely formed in solution above pH
6 and that it is not stable at a pH < 6. When studying the influence of the pH on
the MALDI spectra of Zn-p55F1, we found that the relative intensity of Zn-p55F1
increases with pH. This behavior correlates with the one in solution.
Note that the MALDI spectra at a pH below 6 exhibit Zn-p55F1 complex signals, whereas CD spectra of the solutions at this pH do not. This is most probably
due to the fact that MALDI spectra are taken from crystalline samples. We have
shown in previous work that the amount of complexes present in the solid phase
is higher than in solution if volatile bypro ducts, such as HCl, form upon complexation (Lehmann et al. 1997). This is the case here, because ZnCh was used as
metal salt. The reason for this is that HCl is eliminated during crystallization,
resulting in a shift of equilibrium (2) to the right.
2.2
Zinc Finger Peptide Complexation with Oligodeoxynucleotides
The complexation of Zn-p55F1 to single-stranded nucleic acids is essential for
the replication of HIV-1 and has been extensively studied by various methods in
solution (Lam et al. 1994; South and Summers 1993). In the resulting noncovalent
triple complex the oligodeoxynucleotide binds within a hydrophobic cleft onto
the peptide surface. CCHC zinc fingers bind to ribonucleic acids, but NMR studies in solution, to which our MALDI experiments can be compared, have been
performed using single-stranded oligodeoxynucleotides as structural probes
(South and Summers 1993). Therefore, the MALDI results presented here were
carried out using oligodeoxynucleotides, mainly d(TTGTT).
2-Amino-4-methyl-5-nitropyridin (AMNP) was chosen as a MALDI matrix for
the analysis of the triple complexe (Fitzgerald et al. 1993). This basic matrix
allowed the simultaneous detection of both p55F1 and oligodeoxynucleotide in
positive ion mode. It also permits work at physiological conditions of the sample
solution, using ammonium bicarbonate to adjust the pH.
When analysing the oligodeoxynucleotide-containing samples with MALDI,
undesired alkali ion adducts were first observed. They were eliminated using the
drop dialysis method (Lehmann and Zenobi 1998), where the alkali ions are
exchanged against ammonium ions. Upon sample crystallization, ammonia evaporates and the overall result is that alkali ions are exchanged against protons.
Fig. 9.4A shows the MALDI mass spectrum of a mixture of d(TTGTT) and
p55F1 at neutral pH. The protonated signals of the oligodeoxynucleotide and
p55F1 as well as a small nonspecific adduct of both are detected. Upon Zn 2 + addition, the specific triple complex of p55F1, Zn 2 + and d(TTGTT), which we expect
from solution chemisty, is observed. Less intense p55F1-d(TTGTT) adducts without and with two Zn 2 + ions are also detected. We also succeeded in detecting the
triple complex between p55F1, Zn 2 +, and an oligodeoxynucleotide ll-mer,
d(TTTTTGTTTTT) using a similar sample preparation for MALDI MS (Fig. 9.5).
The signals of both the triple complex and the Zn-p55F1 complex are less
intense than those of the individual components. The reasons in the case of Znp55F1 have already been discussed (Lehmann et al. 1999). Similar arguments can
also be made for the triple complex.
143
ments show that the peptide complex is completely formed in solution above pH
6 and that it is not stable at a pH < 6. When studying the influence of the pH on
the MALDI spectra of Zn-p55F1, we found that the relative intensity of Zn-p55F1
increases with pH. This behavior correlates with the one in solution.
Note that the MALDI spectra at a pH below 6 exhibit Zn-p55F1 complex signals, whereas CD spectra of the solutions at this pH do not. This is most probably
due to the fact that MALDI spectra are taken from crystalline samples. We have
shown in previous work that the amount of complexes present in the solid phase
is higher than in solution if volatile bypro ducts, such as HCl, form upon complexation (Lehmann et al. 1997). This is the case here, because ZnCh was used as
metal salt. The reason for this is that HCl is eliminated during crystallization,
resulting in a shift of equilibrium (2) to the right.
2.2
Zinc Finger Peptide Complexation with Oligodeoxynucleotides
The complexation of Zn-p55F1 to single-stranded nucleic acids is essential for
the replication of HIV-1 and has been extensively studied by various methods in
solution (Lam et al. 1994; South and Summers 1993). In the resulting noncovalent
triple complex the oligodeoxynucleotide binds within a hydrophobic cleft onto
the peptide surface. CCHC zinc fingers bind to ribonucleic acids, but NMR studies in solution, to which our MALDI experiments can be compared, have been
performed using single-stranded oligodeoxynucleotides as structural probes
(South and Summers 1993). Therefore, the MALDI results presented here were
carried out using oligodeoxynucleotides, mainly d(TTGTT).
2-Amino-4-methyl-5-nitropyridin (AMNP) was chosen as a MALDI matrix for
the analysis of the triple complexe (Fitzgerald et al. 1993). This basic matrix
allowed the simultaneous detection of both p55F1 and oligodeoxynucleotide in
positive ion mode. It also permits work at physiological conditions of the sample
solution, using ammonium bicarbonate to adjust the pH.
When analysing the oligodeoxynucleotide-containing samples with MALDI,
undesired alkali ion adducts were first observed. They were eliminated using the
drop dialysis method (Lehmann and Zenobi 1998), where the alkali ions are
exchanged against ammonium ions. Upon sample crystallization, ammonia evaporates and the overall result is that alkali ions are exchanged against protons.
Fig. 9.4A shows the MALDI mass spectrum of a mixture of d(TTGTT) and
p55F1 at neutral pH. The protonated signals of the oligodeoxynucleotide and
p55F1 as well as a small nonspecific adduct of both are detected. Upon Zn 2 + addition, the specific triple complex of p55F1, Zn 2 + and d(TTGTT), which we expect
from solution chemisty, is observed. Less intense p55F1-d(TTGTT) adducts without and with two Zn 2 + ions are also detected. We also succeeded in detecting the
triple complex between p55F1, Zn 2 +, and an oligodeoxynucleotide ll-mer,
d(TTTTTGTTTTT) using a similar sample preparation for MALDI MS (Fig. 9.5).
The signals of both the triple complex and the Zn-p55F1 complex are less
intense than those of the individual components. The reasons in the case of Znp55F1 have already been discussed (Lehmann et al. 1999). Similar arguments can
also be made for the triple complex.