Optimization of protein sample preparation for two-dimensional electrophoresis.
Optimized 2DE sample preparation protocols that maximize total protein recovery are fundamental to improving proteome capture and increasing the utility of 2DE, which is in part limited by inadequate recovery of proteins with diverse physicochemical properties. Maintaining protein solubility is an important factor for protein recovery, but the multitude of solubility-enhancing agents and the relatively low-throughput nature of 2DE limit the systematic study of sample preparation. In this work, design of experiment (DOE) approaches are used to optimize protein recovery by altering the levels of four solubility-enhancing agents (urea, DTT, CHAPS, and SDS) in the initial suspension solution. Protein recovery is quantified by a total protein concentration assay, which is demonstrated to be representative of SDS-PAGE and 2DE recovery. DOE methodologies are presented as relatively high-throughput procedures for optimizing 2DE sample preparation parameters for a variety of sample types. Optimal suspension solution compositions are shown to vary across a model protein solution (no urea or DTT), Chinese hamster ovary (CHO) cell lysate (8 M urea, ≥2% CHAPS, ≥32.5 mM DTT), and Escherichia coli cell lysate (8 M urea, 4% CHAPS, 65 mM DTT), with optimized conditions increasing 2DE protein recovery at least 50% compared to suboptimal conditions.
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware 19711, USA
Kristin N
KN
Valente
Leila H
LH
Choe
Abraham M
AM
Lenhoff
Kelvin H
KH
Lee
D000818:Animals; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D029968:Escherichia coli Proteins; D057166:High-Throughput Screening Assays; D057230:Limit of Detection; D020543:Proteome / Q000032:analysis / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012107:Research Design
D029968:Escherichia coli Proteins; D020543:Proteome