title: OFFGEL fractionation of peptides: where really is your sample?
abstract: Shotgun proteomics of complex samples is generally coupled with at least one peptide fractionation step and, to this effect, peptide isoelectric focusing (IEF) in immobilized pH gradient (IPG) is one of the most used techniques. Fractionation with the OFFGEL 3100 Agilent Technologies apparatus allows the easy recovery of peptides that, after focusing, diffuse into the liquid phase above the gel strip. In this work we investigate the efficiency of peptide diffusion during OFFGEL fractionation and demonstrate that a recovery based only on the spontaneous diffusion process is far from being optimal. We show that a simple additional extraction step with acetonitrile increases of about 40% the amount of material that can be recovered after the focusing. Moreover, we show that the two populations of peptides obtained from the passive elution and from the extraction process are also qualitatively different and only partially overlapping.
authors.affiliation: Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy; Proteomic Center of Padova University, Via G. Orus 2/B, 35129 Padova, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Franchin
authors.affiliation: Proteomic Center of Padova University, Via G. Orus 2/B, 35129 Padova, Italy; Department of Agronomy, Food, Natural Resources, Animals and the Environment, DAFNAE, University of Padova, Viale dell'Università 16, 35020 Legnaro, PD, Italy.
authors.forename: Micaela
authors.initials: M
authors.lastname: Pivato
authors.affiliation: Department of Medicine, University of Padova, Via Giustiniani 2, 35121 Padova, Italy.
authors.forename: Marcello
authors.initials: M
authors.lastname: Rattazzi
authors.affiliation: Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy; Proteomic Center of Padova University, Via G. Orus 2/B, 35129 Padova, Italy. Electronic address: giorgio.arrigoni@unipd.it.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Arrigoni
authors.affiliation: Proteomic Center of Padova University, Via G. Orus 2/B, 35129 Padova, Italy; Department of Medicine, University of Padova, Via Giustiniani 2, 35121 Padova, Italy.
authors.forename: Renato
authors.initials: R
authors.lastname: Millioni
mesh_terms: D005591:Chemical Fractionation / Q000379:methods*; D004058:Diffusion; D010455:Peptides / Q000302:isolation & purification*; D018892:Proton-Motive Force; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D010455:Peptides
keywords: IEF-IPG; OFFGEL Fractionator; Off-gel fractionation; Peptide isoelectrofocusing; Peptide recovery