title: Metal complexes as artificial proteases in proteomics: a palladium(II) complex cleaves various proteins in solutions containing detergents.
abstract: Most popular agents for site-specific protein cleavage are proteolytic enzymes. Because they become denatured and inactivated by detergents, enzymes are inconvenient for proteomic analysis of hydrophobic proteins which require detergents as solubilizing agents. We used cis-[Pd(en)(H(2)O)(2)](2+) (in which en represents ethylenediamine) as an artificial protease to effect cleavage of three bovine proteins, namely ubiquitin, β-casein, and serum albumin, in separate experiments. Cleavage took place in aqueous solutions containing 1.0%wt./vol. of either 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Zwittergent 3-14 at 2.5<pH<2.9 and 55-60°C for 3-72 h. Digests were separated by HPLC and analyzed by tandem mass spectrometry. Peptides were identified by de novo sequencing and matched against the bovine genome. Because cleavage by Pd(II) complexes is rather selective and therefore infrequent, 72% of the identified peptides in the digests contained more than 10 amino acid. Palladium(II) complexes hold promise as cleavage agents in proteomics studies of membrane proteins.
authors: Department of Biological and Environmental Science, Texas A&M University at Commerce, Commerce, TX 75429, USA.
authors: Frank
authors: F
authors: Miskevich
authors: Amy
authors: A
authors: Davis
authors: Porntip
authors: P
authors: Leeprapaiwong
authors: Virginia
authors: V
authors: Giganti
authors: Nenad M
authors: NM
authors: Kostić
authors: Laurence A
authors: LA
authors: Angel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D002793:Cholic Acids / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D056831:Coordination Complexes / Q000737:chemistry* / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D010165:Palladium / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry* / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D002364:Caseins; D002793:Cholic Acids; D056831:Coordination Complexes; D003902:Detergents; D000644:Quaternary Ammonium Compounds; D025801:Ubiquitin; C048256:zwittergent 3-14; D010165:Palladium; D010447:Peptide Hydrolases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate