Investigation of Metalloproteins Utilizing High Resolution Mass Spectrometry

Copper ions (Cu?, Cu??) 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)?Cu?) 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/SO?H) 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/SO?H) are important Cu? ligands. Note that, the proton of the SH/SO?H group is mobile and can be transferred to the arginine guanidino group. For [M + 2Cu - H]? ions, deprotonation of the -SH/SO?H 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.