Proteomics based on peptide fractionation by SDS-free PAGE.
Here we demonstrate the usefulness of peptide fractionation by SDS-free polyacrylamide gel electrophoresis and its applicability to proteomics studies. In the absence of SDS, the driving force for the electrophoretic migration toward the anode is supplied by negatively charged acidic amino acid residues and other residues as phosphate, sulfate and sialic acid, while the resulting mobility depends on both the charge and the molecular mass of the peptides. A straightforward method was achieved for SDS-PAGE of proteins, enzyme digestion, peptide transfer and fractionation by SDS-free PAGE, which was named dual-fractionation polyacrylamide gel electrophoresis (DF-PAGE). This method increases the number of identified proteins 2.5-fold with respect to the proteins identified after direct analysis, and more than 80% of assigned peptides were found in unique SDS-free gel slices. A vast majority of identified peptides (93%) have p I values below 7.0, and 7% have p I values between 7.0 and 7.35. Peptide digests that were derived from complex protein mixtures were in consequence simplified as peptides that are positively charged are not recovered in the present conditions. The analysis of a membrane protein extract from Neisseria meningitidis by this approach allowed the identification of 97 proteins, including low-abundance components.
Ramos
Yassel
Y
Center for Genetic Engineering and Biotechnology, La Habana, Cuba.
Gutierrez
Elain
E
Machado
Yoan
Y
Sánchez
Aniel
A
Castellanos-Serra
Lila
L
González
Luis J
LJ
Fernández-de-Cossio
Jorge
J
Pérez-Riverol
Yasset
Y
Betancourt
Lázaro
L
Gil
Jeovanis
J
Padrón
Gabriel
G
Besada
Vladimir
V
D002364:Caseins / Q000032:analysis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry; D009345:Neisseria meningitidis / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D013300:Streptokinase / Q000032:analysis / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
D002364:Caseins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D013300:Streptokinase; D014357:Trypsin