Recognition of transition metal ions by peptides. Identification of specific metal-binding peptides in proteolytic digest maps by UV laser desorption time-of-flight mass spectrometry.
Metal-binding peptides in proteolytic digest maps have been identified by matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS). The plasma and milk metal transport protein chosen to demonstrate this process, histidine-rich glycoprotein (HRG), was purified and then digested with trypsin; the cleavage products were analyzed by LDTOF-MS with dihydroxybenzoic acid as the matrix. The selective interaction of specific peptides with one or more Cu atoms was observed when Cu(II) ions were added to the digest mixture. At least one specific metal-binding peptide was identified by computerized sequence analysis using the molecular mass data and available cDNA sequence. These results demonstrate the first direct observation by mass spectrometry of differential peptide-metal ion interactions in protein digest maps. The ability to evaluate peptide-metal ion interactions, including stoichiometry, with less than 1 pmol of sample improves significantly our ability to identify metal binding domains in metal-binding proteins.
Hutchens
T W
TW
USDA/ARS Children's Nutrition Research Center, Baylor College of Medicine, Department of Pediatrics, Houston, Texas 77030.
Nelson
R W
RW
Yip
T T
TT
D000595:Amino Acid Sequence; D001665:Binding Sites; D002412:Cations; D002846:Chromatography, Affinity; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007834:Lasers; D013058:Mass Spectrometry / Q000379:methods; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D014357:Trypsin; D000099053:Histidine-Rich Glycoprotein
D002412:Cations; D006023:Glycoproteins; D008670:Metals; D010446:Peptide Fragments; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; D014357:Trypsin