title: Fractionation-dependent improvements in proteome resolution in the mouse hippocampus by IEF LC-MS/MS.
abstract: An assessment of fractionated mouse hippocampal peptides was conducted. Protein extract from a single mouse hippocampus was enzymatically digested and fractionated by IEF. Aliquots of fractions were pooled into fewer, more complex samples. The unfractionated lysate, fractions, and pooled fractions were subjected to LC-MS/MS analysis. Samples consisting of many individual fractions had more protein identifications, greater protein sequence coverage, and quantified proteins with more spectral counts than protein extract that was unfractionated or pooled into fewer LC-MS/MS samples. Additionally, prefractionation reduced the median CV for spectral counts as much as 33%. However, the relative gain in proteome resolution was found to saturate with increasing fractionation extent. This study demonstrates how prefractionation by offline IEF can improve the resolution of proteomic investigations of the mouse hippocampus, and that a data-driven pooling methodology can reduce excessive and cost-ineffective fractionation.
authors.affiliation: Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL, USA.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Bundy
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, FL, USA.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Inouye
authors.affiliation: Translational Science Laboratory, College of Medicine Florida State University, Tallahassee, FL, USA.
authors.forename: Roger S
authors.initials: RS
authors.lastname: Mercer
authors.affiliation: Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL, USA.
authors.forename: Richard S
authors.initials: RS
authors.lastname: Nowakowski
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D020543:Proteome; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D020543:Proteome
keywords: Bioinformatics; Fractionation; IEF; Power analysis; Sample preparation