title: A multidimensional differential proteomic platform using dual-phase ion-exchange chromatography-polyacrylamide gel electrophoresis/reversed-phase liquid chromatography tandem mass spectrometry.
abstract: Differential proteomic analysis has arisen as a large-scale means to discern proteome-wide changes upon treatment, injury, or disease. Tandem protein separation methods are required for large-scale differential proteomic analysis. Here, a novel multidimensional platform for resolving and differentially analyzing complex biological samples is presented. The platform, collectively termed CAX-PAGE/RPLC-MSMS, combines biphasic ion-exchange chromatography with polyacrylamide gel electrophoresis for protein separation, quantification, and differential band targeting, followed by capillary reversed-phase liquid chromatography and data-dependent tandem mass spectrometry for quantitative and qualitative peptide analysis. CAX-PAGE provides high protein resolving power with a theoretical peak capacity of 3570, extendable to 7600, a wide protein mass range verified from 16 to 273 kDa, and reproducible differential sample comparison without the added expense of fluorescent dyes and imaging equipment. Demonstrated using a neuroproteomic model, CAX-PAGE revealed an increased number of differential proteins, 137, compared with 82 found by 2D difference gel electrophoresis. When combined with RPLC-MSMS for protein identification, an additional quantification step is performed for internal validation, confirming a 2-fold or greater change in 89% of identified differential targets.
authors.affiliation: Center for Neuroproteomics and Biomarker Research, University of Florida, Gainesville, Florida 32610, USA. attens@mbi.ufl.edu
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Ottens
authors.forename: Firas H
authors.initials: FH
authors.lastname: Kobeissy
authors.forename: Regina A
authors.initials: RA
authors.lastname: Wolper
authors.forename: William E
authors.initials: WE
authors.lastname: Haskins
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Hayes
authors.forename: Nancy D
authors.initials: ND
authors.lastname: Denslow
authors.forename: Kevin K W
authors.initials: KK
authors.lastname: Wang
mesh_terms: D000818:Animals; D002531:Cerebellum / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D003116:Color; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008297:Male; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*