Proteomic analysis of secreted proteins from Arabidopsis thaliana seedlings: improved recovery following removal of phenolic compounds.
Arabidopsis thaliana seedlings grown in liquid culture were used to recover proteins secreted from the whole plant. The aim was to identify apoplastic proteins that may be lost during classical extraction procedures such as preparation of cell walls. The inclusion of polyvinyl-polypyrrolidone (PVPP) in the protocol of purification of secreted proteins allowed a more efficient identification of proteins after their separation by two-dimensional gel electrophoresis (2-DE) and mass spectrometry analyses. Improvement of identification was 4-fold. It is related to an increased number of detectable peaks on mass spectra increasing the percentage of sequence coverage, and the identification confidence. The role of PVPP was to trap phenolic compounds and to prevent their unspecific interactions with proteins. These experiments resulted in the identification of 44 secreted proteins, of which 70% were not identified in previous cell wall proteomic studies. This may be due to specific gene regulation in seedlings and/or to a better access to apoplastic proteins not bound to cell walls.
Charmont
Stéphane
S
UMR 5546 CNRS-Université Paul Sabatier, Pôle de Biotechnologie Végétale, BP17, 24 chemin de Borde Rouge, 31326 Castanet-Tolosan, France.
Jamet
Elisabeth
E
Pont-Lezica
Rafael
R
Canut
Hervé
H
D017360:Arabidopsis / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D010636:Phenols / Q000737:chemistry* / Q000302:isolation & purification*; D011205:Povidone / Q000031:analogs & derivatives* / Q000494:pharmacology; D040901:Proteomics / Q000379:methods*; D036226:Seedlings / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D029681:Arabidopsis Proteins; D003470:Culture Media; D010636:Phenols; C077842:polyvinylpolypyrrolidone; D011205:Povidone