title: Selective isolation of multiple positively charged peptides for 2-DE-free quantitative proteomics.
abstract: A method for quantitative proteomic analysis based on the selective isolation of multiply charged peptides (RH peptides) containing arginine and histidine residues is described. Two pools of proteins are digested in tandem with lysyl-endopeptidase and trypsin and the primary amino groups of proteolytic peptides are separately labeled with d3- and d0-acetic anhydride. This reaction has a dual purpose: (i) to allow the relative protein quantification in two different conditions and (ii) to restrict the positive charges of peptides to the presence of arginine and histidine. The N-acylated peptides are separated by cation-exchange chromatography into two groups, neutral and singly charged peptides (R+H<or=1) that are neither retained nor analyzed, whereas the multiply charged peptides (R+H>1) are retained into the column and can be eluted in batch or further fractionated using a saline gradient before LC-MS/MS analysis. In silico analysis revealed that the selective isolation of RH peptides considerably simplifies the complex mixture of peptides (three RH peptides/protein) and at the same time they represent 84% of the whole proteomes. The selectivity, and recovery of the method were evaluated with model proteins and with a complex mixture of proteins extracted from Vibrio cholerae.
authors.affiliation: Department of Proteomics, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: Aniel
authors.initials: A
authors.lastname: Sánchez
authors.forename: Luis Javier
authors.initials: LJ
authors.lastname: González
authors.forename: Lázaro
authors.initials: L
authors.lastname: Betancourt
authors.forename: Jeovanis
authors.initials: J
authors.lastname: Gil
authors.forename: Vladimir
authors.initials: V
authors.lastname: Besada
authors.forename: Jorge
authors.initials: J
authors.lastname: Fernández-de-Cossío
authors.forename: Arielis
authors.initials: A
authors.lastname: Rodríguez-Ulloa
authors.forename: Karen
authors.initials: K
authors.lastname: Marrero
authors.forename: Félix
authors.initials: F
authors.lastname: Alvarez
authors.forename: Rafael
authors.initials: R
authors.lastname: Fando
authors.forename: Gabriel
authors.initials: G
authors.lastname: Padrón
mesh_terms: D000089:Acetic Anhydrides / Q000737:chemistry; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D006639:Histidine / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D020543:Proteome / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014734:Vibrio cholerae / Q000737:chemistry*
chemical_list: D000089:Acetic Anhydrides; D010455:Peptides; D020543:Proteome; D011994:Recombinant Proteins; C031800:acetic anhydride; D006639:Histidine; D001120:Arginine; D012697:Serine Endopeptidases; D014357:Trypsin; C036813:lysyl endopeptidase