title: ArgC-Like Digestion: Complementary or Alternative to Tryptic Digestion?
abstract: Enzymatic digestion of complex protein samples is often performed by use of multiple proteases to improve protein identification and characterization. Combining trypsin with ArgC is one option to enhance sequence coverage in bottom-up proteomics. However, the low selectivity of this endoprotease derogates from the benefit of the combination. Our approach here is to mimic ArgC digestion by chemically modifying all lysine residues in proteins so that trypsin can only cleave C-terminal to arginine. Four different amine modifications, dimethylation, acetylation, propionylation, and carbethoxylation, were tested, and the protocols were optimized. A nearly complete conversion of the primary amines was achieved for all modifications. Tryptic digestion of Escherichia coli lysate proteins after acylation of lysine residues shows the most significant improvement compared with data received from ArgC digest. After propionylation, 9216 unique peptides identified 1439 proteins, which, compared with a conventional tryptic digestion, represents the identification of 150 additional proteins due to a reasonable reduction of the sample complexity and higher fragmentation efficiencies of the peptides. It is therefore concluded that the Arg-C like digestion should no longer be regarded as a complementary approach but forms a viable and superior alternative to the conventional trypsin digestion.
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe-University , Frankfurt am Main 60438, Germany.
authors.forename: Vahid
authors.identifier: 0000-0002-8467-6248
authors.initials: V
authors.lastname: Golghalyani
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe-University , Frankfurt am Main 60438, Germany.
authors.forename: Moritz
authors.initials: M
authors.lastname: Neupärtl
authors.affiliation: Functional Proteomics, Centre for Biochemistry, Medical School, Goethe-University , Frankfurt 60590, Germany.|Cluster of Excellence "Macromolecular Complexes", Goethe University , Frankfurt am Main 60438, Germany.|German Center of Cardiovascular Research (DZHK), Partner site RheinMain , Frankfurt 60590, Germany.
authors.forename: Ilka
authors.initials: I
authors.lastname: Wittig
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe-University , Frankfurt am Main 60438, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Bahr
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe-University , Frankfurt am Main 60438, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Karas
mesh_terms: D000107:Acetylation; D000445:Aldehyde Oxidoreductases / Q000737:chemistry*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000737:chemistry; D005019:Ethyl Ethers / Q000737:chemistry; D008745:Methylation; D010446:Peptide Fragments / Q000302:isolation & purification*; D011422:Propionates / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005019:Ethyl Ethers; D010446:Peptide Fragments; D011422:Propionates; D020543:Proteome; D000445:Aldehyde Oxidoreductases; C089846:argC protein, Bacteria; D014357:Trypsin; C029658:propionic acid
keywords: ArgC; acetylation; amino modification; carbethoxylation; on bead digestion; propionylation; protein modification; reductive methylation; shotgun proteomics; trypsin