A multidimensional differential proteomic platform using dual-phase ion-exchange chromatography-polyacrylamide gel electrophoresis/reversed-phase liquid chromatography tandem mass spectrometry.
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.
Ottens
Andrew K
AK
Center for Neuroproteomics and Biomarker Research, University of Florida, Gainesville, Florida 32610, USA. attens@mbi.ufl.edu
Kobeissy
Firas H
FH
Wolper
Regina A
RA
Haskins
William E
WE
Hayes
Ronald L
RL
Denslow
Nancy D
ND
Wang
Kevin K W
KK
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*