Growth and Development of higher plants

Protein identification will be performed in the proteomics and mass spectrometry facilities of P4 which are fully equipped to run various gel electrophoresis systems, liquid chromatography and mass spectrometry analysis.
Protein samples (e.g. different mutants, different developmental stages, different environmental factors) can be compared by 2D gel electrophoresis combining isoelectric fosusing and SDS PAGE. However, this system only reveals the most abundant proteins (circa 600-800 spots). More sensitive approaches can be undertaken by purifying samples, for instance  by subcellular fractionation.
Membrane proteins are poorly resolved by isoelectrocfocusing. In this case, alternative approaches such as cationic/anionic 2D gel electrophoresis or BlueNative/SDS 2D electrophoresis can be used. The latter can also be used to separate under native conditions membrane or soluble complexes and thereafter analyze the subunit composition by SDS gel electrophoresis.
An alternative approach, which avoids any electrophoretic system, consists in trypsinating a protein mix, separating the tryptic fragments by a nano liquid chromatography combining ion exchange and reverse phase and finally submitting the elution fractions to mass spectrometry for peptide identification.
Protein/peptide identification is performed by mass spectrometry, using an ABI 4800 MALDI-TOF/TOF. Identification of Arabidopsis is straightforward since the whole genome is known. Post translational modifications (e.g. phosphorylation, glycosylation) can be identified as well. Enrichment of phosphorylated peptides can be performed by affinity chromatography (ICAT).