Synthetic metal-binding protein surface domains for metal ion-dependent interaction chromatography. I. Analysis of bound metal ions by matrix-assisted UV laser desorption time-of-flight mass spectrometry.
To extend the analytical capabilities of immobilized metal ion affinity chromatography (IMAC) for evaluation of biologically relevant peptide-metal ion interactions, we have prepared synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein known as histidine-rich glycoprotein (HRG). Three synthetic peptides, representing multiples of a 5-residue repeat sequence (Gly-His-His-Pro-His) from within the histidine- and proline-rich region of the C-terminal domain were prepared. Prior to immobilization, the synthetic peptides were evaluated for identity and sample homogeneity by matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS), a method developed recently for the mass determination of high-molecular-mass biopolymers. 2,5-Dihydroxybenzoic acid was evaluated as a matrix to facilitate the laser desorption and ionization of intact peptides and was found to be ideally suited for determinations of mass within the low-mass region of interest (641.7 to 1772.8 dalton). We observed minimal chemical noise from photochemically generated peptide-matrix adduct signals, clustering, and multiply-charged peptide species. Peptides with bound sodium and potassium ions were observed; however, these signal intensities were reduced by immersion of the sample probe tip in water. Mixtures of the three different synthetic peptides were also evaluated by LDTOF-MS after their elution through a special immobilized peptide-metal ion column designed to investigate metal ion transfer. We found LDTOF-MS to be a useful new method to verify the presence of peptide-bound metal ions.(ABSTRACT TRUNCATED AT 250 WORDS)
Hutchens
T W
TW
US Department of Agriculture/Agricultural Research Service, Houston, TX.
Nelson
R W
RW
Li
C M
CM
Yip
T T
TT
D000595:Amino Acid Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D007477:Ions; D007834:Lasers; D013058:Mass Spectrometry / Q000379:methods*; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D014466:Ultraviolet Rays; D000099053:Histidine-Rich Glycoprotein
D001798:Blood Proteins; D007477:Ions; D008670:Metals; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein