High-throughput liquid-liquid fractionation of multiple protein post-translational modifications.
Post-translational protein modifications have contributed significantly to the identification of macromolecular biomarkers of biological processes. We have modified a two-dimensional HPLC system (Beckman Coulter PF2D ProteomeLab) to create proteome maps of post-translational protein modifications. This system resolves complex protein mixtures by anion exchange chromatofocusing in the first dimension and hydrophobicity (reverse phase chromatography) in the second dimension. The simultaneous identification of multiple protein modifications, accomplished by incorporating a photo diode array (PDA) detector into the PF2D system, facilitates the simultaneous production of three-dimensional proteome maps and visualization of both unmodified and post-translationally modified (PTM) proteins at their signature wavelengths within the proteome. We describe procedures for the simultaneous resolution of proteome maps, the identification of proteins modified by nitration, carbonylation, and phosphorylation, and proteins with unique spectra such as the heme containing proteins.
Deford
James H
JH
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-0643, USA.
Nuss
Jonathan E
JE
Amaning
James
J
English
Robert D
RD
Tjernlund
Don
D
Papaconstantinou
John
J
D000818:Animals; D002417:Cattle; D005591:Chemical Fractionation / Q000295:instrumentation / Q000379:methods; D008099:Liver / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry*; D020543:Proteome / Q000032:analysis
D011506:Proteins; D020543:Proteome