title: Improving gel-based proteome analysis of soluble protein extracts by heat prefractionation.
abstract: The presence of high-abundance proteins in complex protein mixtures often masks low-abundance proteins and causes loss of resolution of 2DE. Protein fractionation steps conducted prior to 2DE can enhance the detection of low-abundance proteins and improve the resolution of 2DE. Here, we report a method to prefractionate soluble protein extracts based on protein thermal denaturation. Soluble proteins were extracted from maize embryos and leaves and Escherichia coli cells. Through heating at 95°C for 5 min, soluble protein extracts were prefractionated as heat stable protein fraction (the supernatant) and heat labile protein fraction (the precipitate). Our results showed that heat prefractionation enhanced the separation of proteins in both fractions by 2DE, thereby increasing the chance of detecting low-abundance proteins, many of which were nonvisible in unfractionated extract. In maize embryo, 330 spots were detected in soluble protein extract, while 577 spots were detected after prefractionation. Furthermore, this prefractionation method facilitated the enrichment, detection, and identification of de novo synthesized stress proteins. Because of its simplicity, the one-step heat prefractionation minimizes protein loss. Finally, heat prefractionation requires no expensive special hardware or reagents, and provides an alternative prefractionation for increasing the resolving power of 2DE.
authors.affiliation: Key Laboratory of Physiological Ecology and Genetic Improvement of Food Crops in Henan Province, Henan Agricultural University, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Wu
authors.forename: Erhui
authors.initials: E
authors.lastname: Xiong
authors.forename: Fuju
authors.initials: F
authors.lastname: Tai
mesh_terms: D005591:Chemical Fractionation / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D006358:Hot Temperature; D010940:Plant Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D011489:Protein Denaturation; D055550:Protein Stability; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D012639:Seeds; D012995:Solubility; D003313:Zea mays
chemical_list: D029968:Escherichia coli Proteins; D010940:Plant Proteins; D020543:Proteome