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Selective isolation of multiple positively charged peptides for 2‐DE‐free quantitative proteomics
Author(s) -
Sánchez Aniel,
González López Luis Javier,
Betancourt Lázaro,
Gil Jeovanis,
Besada Vladimir,
FernándezdeCossío Jorge,
RodríguezUlloa Arielis,
Marrero Karen,
Alvarez Félix,
Fando Rafael,
Padrón Gabriel
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200500836
Subject(s) - chemistry , peptide , chromatography , histidine , trypsin , tandem mass spectrometry , proteome , quantitative proteomics , proteomics , acetic anhydride , lysine , arginine , elution , biochemistry , amino acid , mass spectrometry , enzyme , gene , catalysis
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 d 3 ‐ and d 0 ‐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 ≤ 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 .