Metal Affinity Chromatography and Mass Spectrometry*

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 character-ization of metalloproteomes provides valuable informa-tion relating to cellular disposition of metals physiologi-cally 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 SK-Hep-1, which is poorly differentiated. Additionally we studied a single specimen of normal human liver and Hep G2 cells depleted in vitro of cellular copper. We used matrix-assisted laser desorption ionization and electro-spray ionization quadrupole time-of-flight mass spec-trometry to analyze peptide sequences of tryptic digests obtained by either in-gel digestion of metal-binding pro-teins or peptides on an immobilized metal affinity chro-matography column loaded with either Cu or Zn. Mainly high abundance proteins were identified. Cu-binding pro-teins identified included enolase, albumin, transferrin, and alcohol dehydrogenase as well as certain intracellular chaperone proteins. The Cu metalloproteome was not identical to the Zn metalloproteome. Peptide binding ex-periments demonstrated that Cu coordination prefers the order of residues histidine> methionine> cysteine. Al-though the Cu metalloproteome was similar from line to line, subtle differences were apparent. Gel profiling showed more extensive variation in expression of annexin II in SK-Hep-1 and Mz-Hep-1 than in Hep G2 and normal liver tissue. Glycerylphosphorylethanolamine was identi-fied as a post-translational modification at residue Glu-301 of elongation factor 1- in Hep G2. Intracellular copper depletion was associated with loss of the glycerylphos-phoryl side group. These findings suggest that post-translational modification could be affected by intracel-lular actions of copper. Comparison of the Cu and Zn metalloproteomes in Hep G2 with a published general proteome of Hep G2 disclosed little overlap (Seow, T. K.