title: SCX charge state selective separation of tryptic peptides combined with 2D-RP-HPLC allows for detailed proteome mapping.
abstract: Multidimensional peptide fractionation is widely used in proteomics to reduce the complexity of peptide mixtures prior to mass spectrometric analysis. Here, we describe the sequential use of strong cation exchange and reversed phase liquid chromatography in both basic and acidic pH buffers for separating tryptic peptides from complex mixtures of proteins. Strong cation exchange exclusively separates peptide by their charge state into neutral, singly and multi-charged species. To further reduce complexity, each peptide group was separated by reversed phase liquid chromatography at basic pH and the resultant fractions were analyzed by LC-MS/MS. This workflow was applied to a soluble protein lysate from mouse embryonic fibroblast cells, and more than 5000 proteins from 29,843 peptides were identified. The high selectivity displayed during the SCX step (93% to 100%) and the overlaps between proteins identified from the SCX-separated peptide groups, are interesting assets of the procedure.
  
  
  CONCLUSIONS
  The present work shows how complex mixture of peptides can be selectively separated by SCX based essentially on the net charge of peptides. The proposed workflow results in three well-defined subset of peptides of specific amino acid composition, which are representative of the constituent proteins. The very high selectivity obtained (93% to 99%) on the peptide side, underscores for the first time the possibility of SCX chromatography to aid in validating identified peptides.
authors.affiliation: Department of Proteomics, Center for Genetic Engineering and Biotechnology, Ave. 31 e/158 y 190, Cubanacán, Playa, P.O. Box 6162, 10600 Havana, Cuba. Electronic address: lazaro.betancourt@cigb.edu.cu.
authors.forename: Lázaro H
authors.initials: LH
authors.lastname: Betancourt
authors.forename: Pieter-Jan
authors.initials: PJ
authors.lastname: De Bock
authors.forename: An
authors.initials: A
authors.lastname: Staes
authors.forename: Evy
authors.initials: E
authors.lastname: Timmerman
authors.forename: Yasset
authors.initials: Y
authors.lastname: Perez-Riverol
authors.forename: Aniel
authors.initials: A
authors.lastname: Sanchez
authors.forename: Vladimir
authors.initials: V
authors.lastname: Besada
authors.forename: Luis Javier
authors.initials: LJ
authors.lastname: Gonzalez
authors.forename: Joël
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
mesh_terms: D000818:Animals; D002412:Cations; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019295:Computational Biology; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry; D051379:Mice; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D014357:Trypsin / Q000737:chemistry*
chemical_list: D002412:Cations; D010455:Peptides; D014357:Trypsin; D008239:Lysine
keywords: Cation exchange; Proteomics; Reversible alpha-amino group and lysine modification; Selective peptide isolation