Investigation of Metalloproteins Utilizing High Resolution Mass Spectrometry
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Copper ions (Cu⁺, Cu²⁺) play important roles in many biological processes (i.e., oxidation, dioxygen transport, and electron transfer); many of the functions in these processes result from copper ions interacting with proteins and peptides. Previous studies using matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) have shown that Cu⁺ ions preferentially bind to electron rich groups in gas phase (i.e., N-terminal amino group, the side-chains of lysine, histidine and arginine). For peptides with more than one Cu⁺ ligand, the interaction between Cu⁺ ions and ligands is described in terms of competitive binding; however, Cu⁺ coordination chemistry for multiple Cu⁺-containing proteins and peptides in gas phase is still not fully understood. In addition, no studies on the fragmentation chemistry for multiple Cu⁺-binding peptides, such as [M + 2Cu - H]⁺ ions, have been reported. The synthesized dinuclear copper complex (alpha-cyano-4-hydroxycinnamic acid (CHCA) copper salt (CHCA)₄Cu₂) enhances the ion abundances for [M + xCu - (x-1)H]⁺ (x = 1-6) ions in gas-phase when used as a MALDI matrix. Using this matrix we have investigated site-specific copper binding of several peptides using fragmentation chemistry of [M + Cu]⁺ and [M + 2Cu - H]⁺ ions. The fragmentation studies reveal that the binding of a single Cu⁺ ion and two Cu⁺ ions are different, and these differences are explained in terms of intramolecular interactions of the peptide-Cu ionic complex. The competitive Cu⁺ binding to C-terminus (i.e., amide, carboxyl, methyl ester) versus lysine, as well as cysteine (SH/SO₃H) versus arginine (guanidino), was also examined by MALDI MS and theoretical calculations (Density Functional Theory (DFT)). For example, results from theoretical and experimental (fragmentation reactions) studies on [M + Cu]⁺ and [M + 2Cu - H]⁺ ions suggest that cysteine side chains (SH/SO₃H) are important Cu⁺ ligands. Note that, the proton of the SH/SO₃H group is mobile and can be transferred to the arginine guanidino group. For [M + 2Cu - H]⁺ ions, deprotonation of the -SH/SO₃H group is energetically more favorable than that of the carboxyl group, and the resulting thiolate/sulfonate group plays an important role in the coordination structure of [M + 2Cu - H]⁺ ions.
gas phase structure
Wu, Zhaoxiang (2010). Investigation of Metalloproteins Utilizing High Resolution Mass Spectrometry. Doctoral dissertation, Texas A&M University. Available electronically from