Selective isolation of multiple positively charged peptides for 2-DE-free quantitative proteomics

• Published in: Proteomics (Weinheim) Vol. 6; no. 16; pp. 4444 - 4455
• Main Authors: Sánchez, Aniel, González López, Luis Javier, Betancourt, Lázaro, Gil, Jeovanis, Besada, Vladimir, Fernández-de-Cossío, Jorge, Rodríguez-Ulloa, Arielis, Marrero, Karen, Alvarez, Félix, Fando, Rafael, Padrón, Gabriel
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.08.2006; WILEY‐VCH Verlag; Wiley-VCH
• Subjects: Acetic Anhydrides - chemistry; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Arginine - chemistry; Biological and medical sciences; Cation-exchange chromatography; Chromatography, Ion Exchange; Fundamental and applied biological sciences. Psychology; Histidine - chemistry; Mass Spectrometry; Miscellaneous; Molecular Sequence Data; Peptides - analysis; Peptides - chemistry; Protein identification; Proteins; Proteome - chemistry; Recombinant Proteins - analysis; Recombinant Proteins - chemistry; Selective-peptide-isolation; Serine Endopeptidases - metabolism; Trypsin - metabolism; Vibrio cholerae; Vibrio cholerae - chemistry; s spectrometry / Protein identification / Selective-peptide-isolation; Peptides; Proteomics; Isolation; Cations; Identification; Cation-exchange chromatography; Chromatography; Protein; Quantitative analysis
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.200500836

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 ≤ 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.