Peptide-metal ion interactions in solution: Detection by laser desorption time-of-flight mass spectrometry and electrospray ionization mass spectrometry

Abstract

The specific interaction of Cu(I1) ions with metal-binding peptides in solution has been investigated by two different methods of soft ionization mass spectrometry, namely matrix-assisted ultraviolet laser desorption time-of-flight mass spectrometry (LDTOF) and electrospray ionization mass spectrometry (ES). The metal-binding peptide selected for these investigations is a &residue sequence found on the surface of the human plasma metal-transport protein histidine-rich glycoprotein. The peptide, (GHHPH), G, was synthesized and evaluated by ES and LDTOF before and after the addition of Cu(I1) or Mn(I1) ions in solution. In the absence of added metal ions, the peptide was observed to have a mass equal to within 0.5 Da of its calculated mass (2903.0 Da) by both LDTOF and ES. In the presence of CNII), up to five additional peaks were observed at mass increments of approximately 63.9 Da (LDTOF) or 62.3 Da (ES); Mn was not bound to the peptide under identical experimental conditions. By both LDTOF and ES, the maximum Cu-binding capacity observed (i.e., 5 g-atoms mol-') demonstrated that up to 1 Cu could be bound per (GHHPH) internal repeat unit. This peptidemetal ion interaction stoichiometry was verified by direct titration in solution and, with immobilized peptide, by quantitative metal ion affmity chromatography. Thus, the ability to detect stable peptidemetal complexes did not appear to be differentially affected by the two different voIatilization/ionization methods needed to generate charged intact molecular ions. The quantity and stoichiometry of bound Cu atoms was affected, however, by experimental conditions such as LDTOF matrix and ES solution composition. These results demonstrate the ability to verify directly the solution-phase binding capacity of metal-binding peptides by LDTOF and by ES. We conclude from these studies that other metalloorganic interactions may also be amenable to investigation by these rapid and sensitive techniques.

Multiply charged ions

“…These observations are supported by quantum chemical calculations, indicating that cleavage of the amide bond (b-and y-fragments) is favorable only beside a protonated amide nitrogen (166,167). Metal-peptide binding has also been studied (214)(215)(216)(217)(218), most often in the case of Zn2@ and Cu2@ ions that are of particular importance in biochemistry. The ES spectra reflect specific binding of metal to specific regions of the peptide-mainly to histidine residues.…”

Protein fractionation and detection for metalloproteomics: challenges and approaches

“…With regard to metalloproteins (a dedicated review is available elsewhere [90]), many studies concern Ca-binding proteins, for example calmodulin [91], heme-binding proteins [92][93][94], zinc fingers [95][96][97], and metallothioneins [97][98][99][100][101]. The vast majority of these studies used ESI-MS rather than MALDI-MS, although early studies on copper binding to small peptides used both ionisation methods [102].…”

Fragmentation of arginine- and lysine-containing dipeptides cationized by Cu+ and Cu2+

“…Research has also been carried out on a few larger peptides. [19][20][21] Both the number of bound copper atoms and the fragmentation sites seem to be correlated to the presence of high affinity amino acid residues such as histidine.…”