Modeling the isoelectric focusing of peptides in an OFFGEL multicompartment cell.
In proteomic analysis of complex samples at the peptide level (termed shotgun proteomics), an effective prefractionation is crucial to decrease the complexity of the peptide mixture for further analysis. In this perspective, the high-resolving power of the IEF fractionation step is a determining parameter, in order to obtain well-defined fractions and correct information on peptide isoelectric points, to provide an additional filter for protein identification. Here, we explore the resolving power of OFFGEL IEF as a prefractionation tool to separate peptides. By modeling the peak width evolution versus the peptide charge gradient at pI, we demonstrate that for the three proteomes considered in silico (Deinococcus radiodurans, Saccharomyces cerevisiae, and Homo sapiens), 90% of the peptides should be correctly focused and recovered in two wells at most. This result strongly suggests OFFGEL to be used as a powerful fractionation tool in shotgun proteomics. The influence of the height and shape of the compartments is also investigated, to give the optimal cell dimensions for an enhanced peptide recovery and fast focusing time.
Lam
Hoang-Trang
HT
Laboratoire d'Electrochimie Physique et Analytique, Ecole Polytechnique Fédérale de Lausanne, Station 6, CH-1015 Lausanne, Switzerland.
Josserand
Jacques
J
Lion
Niels
N
Girault
H H
HH
D034301:Deinococcus / Q000737:chemistry; D006801:Humans; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods; D008962:Models, Theoretical; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000737:chemistry
D010455:Peptides; D020543:Proteome