Identification of Metal-binding Proteins in Human Hepatoma Lines by Immobilized Metal Affinity Chromatography and Mass Spectrometry

Abstract

The metalloproteome is defined as the set of proteins that have metal-binding capacity by being metalloproteins or having metal-binding sites. A different metalloproteome may exist for each metal. Mass spectrometric characterization of metalloproteomes provides valuable information relating to cellular disposition of metals physiologically and in metal-associated diseases. We examined the Cu and Zn metalloproteomes in three human hepatoma lines: Hep G2 and Mz-Hep-1, which retain many functional characteristics of normal human hepatocytes, and SKHep-1, which is poorly differentiated. Additionally we studied a single specimen of normal human liver and Hep Metals play a pivotal role in cellular metabolism. They function as the catalytic centers in many biochemical reactions and serve as structural elements for a large number of regulatory proteins (1, 2). Characterization of metal-binding proteins is important for understanding the structure and biological functions of such proteins in metal-associated diseases. In the past few years, a variety of mass spectrometric techniques has been used to probe the metal-protein interactions in metal-containing proteins. These studies have been successfully applied to determining metal binding stoichiometry (3-9), metal-binding sites (10, 11), and metal-dependent structure/conformation changes (12,13). A number of metalbinding proteins, such as cytochrome c oxidase (14), albumin (3, 7), metallothionein (12,15,16), prion protein (PrP) (8,9,11,13,17), matrilysin (4, 5), and non-heme iron-containing metalloproteins (6), have been individually well characterized by either overall mass measurements on the intact metal-protein complexes or peptide sequencing on the protein digest with tandem mass spectrometry.Rapid developments in proteomic technology and bioinformatics have permitted identification of proteomes of cell lines and tissue by using mass spectrometry instrumentation (for review, see . The specific advances include the use of two-dimensional gel electrophoresis (2DE),

Divalent Metal Ions in Plant Mitochondria and Their Role in Interactions with Proteins and Oxidative Stress-Induced Damage to Respiratory Function

“…Like the IMAC study of Kung et al (2006), we have found that IMAC selects subsets of proteins that contain significant numbers of putative metal interaction motifs on their surfaces. In the metal-protein interaction studies of Cu ions and liver disease (She et al, 2003;Smith et al, 2004), nickel hypersensitivity in human B cells (Heiss et al, 2005), and Cu ion homeostasis in Arabidopsis roots (Kung et al, 2006), no attempt was made to localize the metal-binding motifs on homologous proteins. However, the surface localization of the putative metal-binding motifs is supported by reports of the use of IMAC to assist in protein crystallization through surface His residues (Frey et al, 1996) and reports of protein contaminants in metal affinity purification of His-tagged recombinant proteins due to natural surface metal-binding motifs (Cai et al, 2004;Bolanos-Garcia and Davies, 2006).…”

Liver as a key organ in the supply, storage, and excretion of copper

“…Some of the proteins of interest were known to have copper-binding capability, but others were not previously known to be able to bind copper. We also examined copper depletion and found a novel variation in posttranslational modification of elongation factor 1␣, depending on whether HepG2 cells were copper depleted (20). A more focused examination of HepG2 cytosol and microsomes showed a broader spectrum of copper-binding proteins (21).…”

Metallomics: Guidelines for terminology and critical evaluation of analytical chemistry approaches (IUPAC Technical Report)

“…The metal-coordination center is expected to be preserved by the retention of the metal-peptide complex after the digestion of the protein [56,57], which seems to be a convenient way for the identification of the metal-binding motives…”

“…The IMAC approach enjoys a growing popularity with recent applications referring to the analysis of the zinc [56,57] and copper [56][57][58] proteomes in human hepatoma cell lines, uranyl proteome in human serum [59], bismuth proteome in Helicobacter pylori cell extracts [60], and Ni-proteome in human keratinocytes [61,62] and Arabidopsis thaliana roots [63].…”