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Immobilized Metal Ion Interaction 27
I-
r
500
J
GHHPHG (1 -mu)
10
L
I 2
21
GHHPHGHHPHGHHPHG (3-mer)
1000 1500 2000
m/z
Fig. 5. Matrix-assisted UV laser desorption time-of-flight mass spec-
trometry (LDTOF) of a mixture of three synthetic metal-binding peptides
(1-mer, 2-mer, and 3-mer) after elution from a column of immobihzed
GHHPHGHHPHG (2-mer) loaded with CuW) ions (14). The synthetic pep-
tide-metal ion affinity column used for metal ion transfer was prepared by
coupling GHHPHGHHPHG (2-mer) to Affi-10 (Bio-Rad). CU(I1) ions were
loaded as described in Section 3.1. The column was equilibrated with 20
mM sodium phosphate buffer (pH 7.0) with 0.5M NaCl. An equimolar mix-
ture of the three different synthetic peptides (free of bound metal ions)
was passed through the column unretained. Flow-through peptide frac-
tions were anaylzed directly by LDTOF (23-25). The metal ion-free pep-
tides GHHPHG (1-mer peak l.O), GHHPHGHHPHG (2-mer peak 2.0), and
GHHPHGHHPHGHHPHG (3-mer peak 3.0) are observed along with pep-
tides with 1, 2,3, or 4 bound Cu(II) ions (e.g., 3.1,3.2., 3.3 ,3.4). The small
peaks marked by an asterisk indicate the presence of a peptide-sodium
adduct ion. A detailed description of these results IS provided in ref. 14
(reproduced with permission).
2. The phosphate buffer pH gradient is good for the pH ‘7-4.5 range. It
has the advantage of UV transparency and is particularly suitable for
peptide analysis (17).
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