title: Evaluation of different multidimensional LC-MS/MS pipelines for isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis of potato tubers in response to cold storage.
abstract: Cold-induced sweetening in potato tubers is a costly problem for the food industry. To systematically identify the proteins associated with this process, we employed a comparative proteomics approach using isobaric, stable isotope coded labels to compare the proteomes of potato tubers after 0 and 5 months of storage at 5 °C. We evaluated both high pH reverse phase (hpRP) liquid chromatography (LC) and off-gel electrophoresis (OGE) as first dimension fractionation methods followed by nanoLC-MS/MS, using two high performance mass spectrometry platforms (Q-TOF and Orbitrap). We found that hpRP-LC consistently offered better resolution, reduced expression ratio compression, and a more MS-compatible workflow than OGE and consistently yielded more unique peptide/protein identifications and higher sequence coverage with better quantification. In this study, a total of 4463 potato proteins were identified, of which 46 showed differential expressions during potato tuber cold storage. Several key proteins important in controlling starch-sugar conversion, which leads to cold-induced sweetening, as well as other proteins that are potentially involved in this process, were identified. Our results suggest that the hpRP-RP shotgun approach is a feasible and practical workflow for discovering potential protein candidates in plant proteomic analysis.
authors.affiliation: Robert W. Holley Center for Agriculture and Health, USDA-ARS, Cornell University , Ithaca, New York 14853, United States. yy44@cornell.edu
authors.forename: Yong
authors.initials: Y
authors.lastname: Yang
authors.forename: Xu
authors.initials: X
authors.lastname: Qiang
authors.forename: Katherine
authors.initials: K
authors.lastname: Owsiany
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhang
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Thannhauser
authors.forename: Li
authors.initials: L
authors.lastname: Li
mesh_terms: D050260:Carbohydrate Metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D015925:Cryopreservation; D005511:Food Handling / Q000379:methods; D005519:Food Preservation / Q000379:methods; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D011198:Solanum tuberosum / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D010455:Peptides; D010940:Plant Proteins