Muth-Pawlak - SRM_irondepr_2015

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Development of a quantitative SRM-based proteomics method to study iron metabolism of Synechocystis sp. PCC 6803

  • Organism: Synechocystis sp. PCC 6803
  • Instrument: TSQ Vantage
  • SpikeIn: No
The cyanobacterium Synechocystis sp. PCC 6803 is a well-established model species in oxygenic photosynthesis research, and a potential host for biotechnological applications. Despite recent advances in genome sequencing and microarray techniques applied in systems biology, quantitative proteomics approaches with corresponding accuracy and depth are scarce for S. 6803. In this study we developed a protocol to screen changes in the expression of 106 proteins representing central metabolic pathways in S. 6803 with targeted mass spectrometry method, selected reaction monitoring (SRM). We evaluated the response to the exposure of both short and long term iron deprivation. The experimental setup enabled the relative quantification of 96 proteins, with 87 and 92 proteins showing adjusted p-values <0.01 under short and long term iron deficiency, respectively. The high sensitivity of the SRM method for S. 6803 was demonstrated by providing quantitative data for altogether 64 proteins that previously could not be detected with the classical data-dependent MS approach under similar conditions. This highlights the effectiveness of SRM for quantification and extends the analytical capability to low abundant proteins in unfractionated samples of S. 6803. The SRM assays and other generated information are now publicly available via PASSEL and Panorama.
Experiment Description
The target Synechocystis sp. PCC 6803 strain was cultivated under the iron sufficient and deprived (harvested once the OD750nm reached 1.0 and after 12 days) conditions. The proteins were extracted and digested and the peptides mixtures were subjected to shotgun LC-MS/MS analysis (QExactive and Orbitrap-FT mass spectrometers). The obtained data was searched against S. 6803 proteome and the combined results led to discovery of altogether 1710 proteins, represented in total by 15031 unique peptides (46 % coverage of the S. 6803 proteome). SRM-assays were developped for 106 proteins covering three functional categories : (1) photosynthesis and carbon fixation, (2) Other anabolic reactions/ auxiliary metabolism and (3) catabolism/ amphibolic pathways. We were able to reliably detect (TSQ Vantage) and quantify 96 proteins in unfractionated cell lysates, with 87 and 92 proteins showing p-values <0.01 under short and long term iron deficiency, respectively. Moreover 64 proteins have never been quantified in similar experimental conditions.
Sample Description
Sample preparation Synechocystis sp. PCC 6803 was grown in BG11 medium, pH 8.0, at +30 °C under a light intensity of approximately 50 μmol photons m− 2 s− 1 in an AlgaeTron230 incubator under agitation of 150 rpm under 1 % (v/v) CO2. For the iron depletion the cells were grown in BG11-medium, where FeNH4-citrate was eliminated. The cell density was measured on Genesys 10S UV-VIS spectrophotometer (Thermo Scientific). The precultures were grown under normal conditions in normal BG11, pH 8.0, in an incubator filled with 1% CO2. The cell growth was monitored by measuring the optical density at 750 nm (OD750nm) over time and the cells were collected by centrifugation (4000 x g 10 minutes at room temperature) once OD750nm reached ~1.0. The cells were washed twice either with iron sufficient or depleted BG11, depending of the future treatment, and centrifuged as previously described. The washed precultures were inoculated to 50 ml batches of respective growth media and the starting OD750nm was adjusted to ~0.1. In order to remove all traces of iron from the culture flasks, the glassware were treated with 10 % HCl and 10 µM EDTA prior autoclaving. The cell growth was measured over time and the cells were harvested from both iron sufficient and deprived conditions, once the OD750nm reached 1.0 and after 12 days under iron deprivation. The growth patterns were verified by repeating the growth experiments several times. The cells were collected by centrifugationand washed twice with 50 mM TES-KOH buffer, pH 8.0. The cell pellet was suspended in protein extraction buffer (0.1 % (w/v) RapiGest SF in 8 M urea solubilized in 0.1 M NH4HCO3 supplemented with 200 µM PMSF) with equal volume of acid washed glass beads (150-212 µm, Sigma).The cells were broken by bead beater and unbroken cells and glass beads were removed by centrifugation. The proteins were reduced with dithiotreitol and alkylated with iodoacetamide. The protein extracts were precipitated with 1:5 v/v of 50% acetone/ 50% ethanol o/n at -20 °C. The pellet was solubilized by trypsin digestion (1:100 trypsin:protein ratio) in 50 mM NH4HCO3 and 5 % acetonitrile (ACN) buffer for 4-5 hours at +37 °C shaking. Digestion was continued for an additional 15-16 hours following a second addition of trypsin in the same ratio. The digestion was stopped by adding formic acid (FA) (Sigma) to a final concentration of 0.5–1 % to lower the pH below 2. The digestion mixture was incubated for 30 min at +37 °C at 130 rpm and centrifuged. To desalt the samples, solid phase extraction (SPE) of the peptide mixture was performed with 4 mm/1 ml extraction disk cartridge (Empore™ C18-SD, 3M™) according to manufactures protocol. The eluted peptide samples were vacuum-dried (Savant SPD1010, SpeedVac Concentrator, Thermo Scientific) and solubilized in 0,1 % FA and 2 % ACN and stored at -80 °C prior to MS analysis.
Created on 8/10/15 12:58 PM