Intact mass measurements for unequivocal identification of hydrophobic photosynthetic photosystems I and II antenna proteins.
The purpose of this study was to determine the optimal experimental conditions necessary to allow rapid and accurate identification of highly hydrophobic proteins, such as the antenna proteins from photosystems I and II. The antenna proteins were derived from two different species, tomato and Arabidopsis, whose photosynthetic genome is well known. The separation techniques included sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting, microamino acid sequencing, reversed-phase high-performance liquid chromatography (RP-HPLC), intact mass measurements, and peptide mass fingerprinting by mass spectrometry. Immunoblotting was time-consuming, and success was limited due to cross-reactivity between these highly conserved sequences. The best results by far were achieved by separating intact proteins through hyphenation of reversed-phase liquid chromatography on-line to an electrospray ionization mass spectrometer (ESI-MS) and consequently identifying individual proteins from their intact mass measurements (IMMs), whereas peptide mass fingerprinting was hampered by the highly hydrophobic nature of these proteins. RP-HPLC-ESI-MS was superior in the quality of separation. Moreover, the high quality of mass spectra recorded during the RP-HPLC-ESI-MS analysis meant that the relative deviations of the molecular masses determined with a quadrupole ion trap mass analyzer ranged between 100 and 300 ppm. Thus, the correspondence between the intact mass values measured with those deduced from the DNA sequences allowed the different types of antenna proteins to be identified and assigned to their corresponding gene families. By utilizing this correlation, it was possible to spot gene products of previously cloned genes.
Zolla
Lello
L
Department of Environmental Sciences, University of Tuscia, Viterbo, Italy. zolla@unitus.it
Rinalducci
Sara
S
Timperio
Anna Maria
AM
Huber
Christian G
CG
Righetti
Pier Giorgio
PG
D017360:Arabidopsis / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D057927:Hydrophobic and Hydrophilic Interactions; D018551:Solanum lycopersicum / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods; D045322:Photosynthetic Reaction Center Complex Proteins / Q000032:analysis*; D045331:Photosystem I Protein Complex / Q000737:chemistry*; D045332:Photosystem II Protein Complex / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D045332:Photosystem II Protein Complex