title: Development of an integrated approach for evaluation of 2-D gel image analysis: impact of multiple proteins in single spots on comparative proteomics in conventional 2-D gel/MALDI workflow.
abstract: With 2-D gel mapping, it is often observed that essentially identical proteins migrate to different positions in the gel, while some seemingly well-resolved protein spots consist of multiple proteins. These observations can undermine the validity of gel-based comparative proteomic studies. Through a comparison of protein identifications using direct MALDI-TOF/TOF and LC-ESI-MS/MS analyses of 2-D gel separated proteins from cauliflower florets, we have developed an integrated approach to improve the accuracy and reliability of comparative 2-D electrophoresis. From 46 spots of interest, we identified 51 proteins by MALDI-TOF/TOF analysis and 108 proteins by LC-ESI-MS/MS. The results indicate that 75% of the analyzed spots contained multiple proteins. A comparison of hit rank for protein identifications showed that 37 out of 43 spots identified by MALDI matched the top-ranked hit from the ESI-MS/MS. By using the exponentially modified protein abundance index (emPAI) to determine the abundance of the individual component proteins for the spots containing multiple proteins, we found that the top-hit proteins from 40 out of 43 spots identified by MALDI matched the most abundant proteins determined by LC-MS/MS. Furthermore, our 2-D-GeLC-MS/MS results show that the top-hit proteins in 44 identified spots contributed on average 81% of the spots' staining intensity. This is the first quantitative measurement of the average rate of false assignment for direct MALDI analysis of 2-D gel spots using a new integrated workflow (2-D gel imaging, "2-D GeLC-MS/MS", and emPAI analysis). Here, the new approach is proposed as an alternative to traditional gel-based quantitative proteomics studies.
authors.affiliation: US Plant, Soil and Nutrition Laboratory, USDA-ARS, Ithaca, NY, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Yang
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Thannhauser
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D001937:Brassica / Q000737:chemistry; D002853:Chromatography, Liquid; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D007202:Indicators and Reagents; D008970:Molecular Weight; D010449:Peptide Mapping; D010940:Plant Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D007202:Indicators and Reagents; D010940:Plant Proteins