LC-MS/MS methods for absolute quantification and identification of proteins associated with chimeric plant oil bodies.
Oil bodies (OBs) are plant cell organelles that consist of a lipid core surrounded by a phospholipid monolayer embedded with specialized proteins such as oleosins. Recombinant proteins expressed in plants can be targeted to OBs as fusions with oleosin. This expression strategy is attractive because OBs are easily enriched and purified from other cellular components, based on their unique physicochemical properties. For recombinant OBs to be a potential therapeutic agent in biomedical applications, it is necessary to comprehensively analyze and quantify both endogenous and heterologously expressed OB proteins. In this study, a mass spectrometry (MS)-based method was developed to accurately quantify an OB-targeted heterologously expressed fusion protein that has potential as a therapeutic agent. The effect of the chimeric oleosin expression upon the OB proteome in transgenic plants was also investigated, and the identification of new potential OB residents was pursued through a variety of liquid chromatography (LC)-MS/MS approaches. The results showed that the accumulation of the fusion protein on OBs was low. Moreover, no significant differences in the accumulation of OB proteins were revealed between transgenic and wild-type seeds. The identification of five new putative components of OB proteome was also reported.
Laboratorio Biotecnologie Unità Tecnica BIORAD, ENEA CR Casaccia, Roma, Italy.
Floriana
F
Capuano
Nicholas J
NJ
Bond
Laurent
L
Gatto
Frédéric
F
Beaudoin
Johnathan A
JA
Napier
Eugenio
E
Benvenuto
Kathryn S
KS
Lilley
Selene
S
Baschieri
D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D010938:Plant Oils / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis* / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D029681:Arabidopsis Proteins; D010938:Plant Oils; D010940:Plant Proteins; D011994:Recombinant Proteins