Characterization by ¹⁵N-metabolic labeling and mass spectrometry of proteins for high-light adaptations in Synechococcus sp. PCC 7002 cyanobacteria
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