A proteomic approach for investigation of photosynthetic apparatus in plants.
The proteome of the photosynthetic apparatus of barley (Hordeum vulgare), obtained by analysis of thylakoids without any previous fractionation, was mapped by native electrophoresis followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as the second dimension two-dimensional-blue native (2-D/BN)/SDS-PAGE). This protocol provided an excellent alternative to the 2-D-isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 2-D separation of the most hydrophobic thylakoid proteins. Monocots and dicots showed significant differences in the first dimension while in the second dimension patterns appeared similar. Identification of each spot was performed by internal peptide primary sequence determination using both nano-electrospray ionization tandem mass spectrometry and, to a lesser extent, peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight using MALDI-TOF. This is due in particular to the fact that a limited number of peptides was obtained after trypsin digestion of these highly hydrophobic proteins. A larger number of peptides from hydrophilic intermembrane domains of transmembrane proteins were detected. Despite this, about 70% of the expected proteins were identified, including proteins with grand average of hydropathicity scores higher than 0.5. It is therefore reasonable to assert that protein hydrophobicity is not the limiting factor. Small proteins were not well identified with trypsin digestion. Instead some of these could be identified using acid hydrolysis. The method presented here does not require prefractionation of different thylakoid complexes and consequently gives confidence in comparing the proteome of the photosynthetic apparatus before and after treatment. It thus allows us to understand the molecular mechanisms underlying physiological adaptations of higher plants and to perform screening of photosynthetic mutants.
Ciambella
Corrado
C
Department of Environmental Sciences, University of Tuscia, Viterbo, Italy.
Roepstorff
Peter
P
Aro
Eva Mari
EM
Zolla
Lello
L
D002734:Chlorophyll / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D001467:Hordeum / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D010449:Peptide Mapping; D045322:Photosynthetic Reaction Center Complex Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020524:Thylakoids / Q000737:chemistry
D045322:Photosynthetic Reaction Center Complex Proteins; D020543:Proteome; D002734:Chlorophyll