Determination of calcium binding sites in gas-phase small peptides by tandem mass spectrometry.
Low-energy (LE) and high-energy (HE) collisionally activated decompositions (CAD) of calcium/peptide complexes of the form [M - H + Ca]+ and [M + Ca]2+ reflect the site of calcium binding in various gas-phase peptides that are models of the calcium binding site III of rabbit skeletal troponin C. The Ca2+ binding sites involve an aspartic acid, glutamic acid, and asparagine, which are in the metal-binding loops of calcium-binding proteins. Both fast atom bombardment (FAB) and electrospray ionization (ESI) were used to generate the metal/peptide complexes. When submitted to LE CAD, ESI-produced Ca2+/peptide complexes undergo fragmentations that are controlled by Ca2+ binding and provide information on the Ca2+ binding site. The LE CAD spectra are simple, indicating that Ca2+ binding involves specific oxygen ligands including acidic side chains and that only a few low-energy fragmentation channels exist. The HE CAD spectra of FAB-produced Ca2+/peptide complexes are more complex, owing to the introduction of high internal energy into the precursor ion. Interactions of the other alkaline-earth metal ions Mg2+ and Ba2+ with these peptides reveal that the ligand preferences of these metal ions are slightly different than those of Ca2+.
Chemistry Department, Washington University, St. Louis, MO 63130, USA.
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Nemirovskiy
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Gross
D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D005740:Gases; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry*; D016339:Spectrometry, Mass, Fast Atom Bombardment
D002135:Calcium-Binding Proteins; D005740:Gases; D007202:Indicators and Reagents; D010455:Peptides