title: A comparison of protein extraction methods suitable for gel-based proteomic studies of aphid proteins.
abstract: Protein extraction methods can vary widely in reproducibility and in representation of the total proteome, yet there are limited data comparing protein isolation methods. The methodical comparison of protein isolation methods is the first critical step for proteomic studies. To address this, we compared three methods for isolation, purification, and solubilization of insect proteins. The aphid Schizaphis graminum, an agricultural pest, was the source of insect tissue. Proteins were extracted using TCA in acetone (TCA-acetone), phenol, or multi-detergents in a chaotrope solution. Extracted proteins were solubilized in a multiple chaotrope solution and examined using 1-D and 2-D electrophoresis and compared directly using 2-D Difference Gel Electrophoresis (2-D DIGE). Mass spectrometry was used to identify proteins from each extraction type. We were unable to ascribe the differences in the proteins extracted to particular physical characteristics, cell location, or biological function. The TCA-acetone extraction yielded the greatest amount of protein from aphid tissues. Each extraction method isolated a unique subset of the aphid proteome. The TCA-acetone method was explored further for its quantitative reliability using 2-D DIGE. Principal component analysis showed that little of the variation in the data was a result of technical issues, thus demonstrating that the TCA-acetone extraction is a reliable method for preparing aphid proteins for a quantitative proteomics experiment. These data suggest that although the TCA-acetone method is a suitable method for quantitative aphid proteomics, a combination of extraction approaches is recommended for increasing proteome coverage when using gel-based separation techniques.
authors.affiliation: USDA, ARS, Robert W. Holley Center for Agriculture and Health, Department of Plant Breeding and Genetics, Cornell University, Ithaca, NY 14853, USA.
authors.forename: M
authors.initials: M
authors.lastname: Cilia
authors.forename: T
authors.initials: T
authors.lastname: Fish
authors.forename: X
authors.initials: X
authors.lastname: Yang
authors.forename: M
authors.initials: M
authors.lastname: McLaughlin
authors.forename: T W
authors.initials: TW
authors.lastname: Thannhauser
authors.forename: S
authors.initials: S
authors.lastname: Gray
mesh_terms: D000096:Acetone; D000818:Animals; D001042:Aphids / Q000737:chemistry*; D002232:Carbocyanines / Q000032:analysis; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005260:Female; D005456:Fluorescent Dyes / Q000032:analysis; D019476:Insect Proteins / Q000032:analysis / Q000302:isolation & purification*; D013058:Mass Spectrometry; D019800:Phenol; D040901:Proteomics / Q000379:methods*; D014020:Tissue Extracts / Q000032:analysis; D014238:Trichloroacetic Acid
chemical_list: D002232:Carbocyanines; D003902:Detergents; D005456:Fluorescent Dyes; D019476:Insect Proteins; D014020:Tissue Extracts; D000096:Acetone; D019800:Phenol; D014238:Trichloroacetic Acid