LC-MSMS of RIBOPROTEOMES OF HEALTHY, POTATO VIRUS A- AND AGROBACTERIUM -INFECTED NICOTIANA BENTHAMIANA PLANTS
Nicotiana benthamiana is an important model plant for plant-microbe interaction studies. We compared the proteomes of ribosomes purified from healthy N. benthamiana plants and plants that were infected with two plant pathogens potato virus A (PVA genus Potyvirus) and A. tumefaciens bacteria. Ribosomes were affinity purified from transgenic leaves that expressed FLAG-tagged RPL18B (RPL: ribosome protein large subunit) of A. thaliana. Control purifications were made from non-transgenic plants that were infected with PVA. Our riboproteome revealed ~6600 r-protein hits representing 424 distinct r-proteins that were members from 71 of the expected 81 r-protein families.
Sample Processing Protocol
Four independent plant batches were infiltrated with A. tumefaciens carrying 35S-fluc-nos binary expression construct or A. tumefaciens harboring an infectious cDNA (icDNA) clone of PVA. A subset of plants was left untreated to represent healthy plants. In addition non-transgenic plants were infected with PVA to serve as control for non-specific purification. Ribosomes were affinity purified using ANTI-FLAG M2 affinity gel beads from leaves that were collected 3 and 4 days post infection. Frozen, pulverized leaf tissue (~4 ml) was homogenized with one volume of polysome extraction buffer [PEB: 200 mM Tris-HCl (pH 9.0), 200 mM KCl, 36 mM MgCl2, 10 mM EGTA, 1 mg/ml heparin, 1 mM DTT, 50 µg/ml cycloheximide, 50 µg/ml chloramphenicol, 2% Triton X-100, 2% Tween 40, 2% Brij35, 2% NP-40, 2% PTE, 1% deoxycholine] for 30 min at 4⁰C. Homogenates were clarified by two subsequent centrifugations at 16 000 x g for 10 min at 4⁰C. Equal amounts of lysates were mixed with 50 µl of ANTI-FLAG M2 affinity gel beads (Sigma-Aldrich, USA) at 4⁰C with gentle rotation for 1 h. After incubation, cleared resin was briefly rinsed with 1 ml of PEB, followed by incubation with PEB for 5 min at 4⁰C. Next, the resin was washed three times for 5 min with 1 ml of washing buffer [WB: 40 mM Tris-HCl (pH 8.8), 100 mM KCl, 10 mM MgCl2] at 4⁰C. Ribosomes were eluted with WB that contained 200 ng/ml of 3xFLAG peptide (Sigma-Aldrich, USA) at 4⁰C for 30 min. Eluted material was stored at -70⁰C. Ribosome samples were digested by trypsin and peptides were purified with C18 microspin columns and re-dissolved in 30 µl of buffer A (0.1% trifluoroacetic acid and 1% acetonitrile in MS grade water). Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis was carried out on an EASY-nLC (Thermo Fisher Scientific, Germany) connected to a Velos Pro-Orbitrap Elite ETD hybrid mass spectrometer (Thermo Fisher Scientific, Germany) with nano electrospray ion source (Thermo Fisher Scientific, Germany). The LC-MS/MS samples were separated using a two-column setup consisting of on a 2 cm C18-A1 trap column (Thermo Fisher Scientific, Germany), followed by a 10 cm C18-A2 analytical column (Thermo Fisher Scientific, Germany). Each sample was analyzed in duplicate. The analyses were performed in a data-dependent acquisition mode using collision-induced dissociation (CID). Full MS scan was acquired with a resolution of 60 000 at normal mass range in the orbitrap analyzer. The method was set to fragment the 20 most intense precursor ions with CID (energy 35).
Data Processing Protocol
Data was acquired using LTQ Tune software (Thermo Fisher Scientific, Germany). The calibrated peak files from the Orbitrap Elite were searched against the N. benthamiana protein sequence database (Niben.genome.v0.4.4.proteins.annotated.fasta) downloaded from the Sol Genomics Network (www.solgenomics.net) using SEQUEST. The fasta file was modified to include the PVA-encoded proteins P1, HCPro, 6K1, CI, 6K2, VPg, NIa, NIb, Rluc, and CP. Bacterial protein sequences were obtained from the complete sequence of A. tumefaciens str. C58 circular chromosome (accession numbers AE007869, AE007943-AE008196, AE008688 and AE008975-AE009230) (Wood et al., Science 294:2317-2323). Error tolerances on the precursor and fragment ions were ±15 ppm and ±0.6 Da, respectively. Database searches were performed to tryptic peptides allowing maximum of two missed cleavages. Carbamidomethyl cysteine and methionine oxidation or phosphorylation (S/T/Y) were set as fixed and variable modifications, respectively. For peptide identification the FDR < 0.05 was used. Proteins with peptide matching score (PSM) equal or above two were included in the analysis. Whole riboproteome data was also used to identify potential phosphoproteins. Outputs of the individual runs (four treatments, four purifications, two technical repetitions) were merged and filtered in Excel (Supplementary Fig. X). First proteins that were identified with PSM value equal to one were removed. Next, proteins that were identified in the non-transgenic control samples were removed from the FLAG-RPL18 samples to exclude non-specific binders from the analysis. The number of distinct proteins was obtained by removing multiple hits. Proteins were divided to ribosomal proteins and potential ribosome-associated proteins based on the annotations. Ribosomal proteins were further subdivided to RPS- and RPL-proteins. Paralogs were identified based on unique peptides.
Eskelin K, Varjosalo M, Ravantti J, Mäkinen K. Ribosome profiles and riboproteomes of healthy and potato virus A and Agrobacterium -infected Nicotiana benthamiana plants. Mol Plant Pathol. 2018 PubMed: 30375150