Characterization by ¹⁵N-metabolic labeling and mass spectrometry of proteins for high-light adaptations in Synechococcus sp. PCC 7002 cyanobacteria
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The marine cyanobacterium Synechococcus sp. PCC 7002 tolerates extreme highlight intensities of more than twice full sunlight (≥2000 μmol photons m-2 s-1), a stress condition that can damage cellular machinery via the production of reactive oxygen species (ROS). While many cyanobacterial photoprotective mechanisms have been identified to mitigate ROS-induced damage, quantification of the protein response of Synechococcus sp. PCC 7002 to extreme high light intensity may contribute to our understanding of these processes. 14N- and 15N-metabolic labeling of Synechococcus sp. PCC 7002 cultures, coupled with electrospray (ESI)-Ion trap and MALDI-TOF mass spectrometry (MS) analyses of the tryptic peptides, were employed to quantify protein levels in response to high- versus optimal-light intensity. The ESI-Ion trap mass spectrometer provided tandem, fragmentation (MS2) mass/charge (m/z) lists that were searched against a custom MASCOT database to identify the 14N and 15N peptide sequences. Optimization of the ESI-Ion trap MS parameters increased the total number of compounds and protein identifications by ~2-fold. Separation of the soluble protein fraction into SDS-PAGE gel-band digests resulted in a similar number of ESI-Ion trap MS protein identifications (IDs) as the bulk digest (29 vs 28). However, the separate gelband MS analyses resulted in 19 additional unique protein IDs. Both 14N-high- and 15N-optimal-light peptides were detected by ESI-Ion trap MS, and showed characteristic m/z mass shifting due to the 14N (light) versus 15N (heavy) isotopic incorporation. Some of these 14N-high- and 15N-optimal-light peptides were cross-detected by MALDI-TOF mass spectrometry for relative protein quantification analyses. Phycobilisome light harvesting proteins and membrane-fraction photosystem protein were predominantly detected, and levels decreased 2-fold under high-light intensity. Work presented here demonstrate that downregulation of light-harvesting and photosynthesis proteins is part of the adaptive mechanism that allows Synechococcus sp. PCC 7002 to thrive at high light intensities. Furthermore, 15N-metabolic labeling of Synechococcus 7002 cells by substituting 15N-NaNO3 in the growth medium in place of 14N-NaNO3 is a simple and effective strategy for isotopic labeling of proteins for quantitative mass spectrometry.
Cyanobacteria, Effect of light on
"A Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science-Biology Microbiology "