Plant SILAC: Stable-Isotope Labelling with Amino Acids of Arabidopsis Seedlings for Quantitative Proteomics.
Stable Isotope Labelling by Amino acids in Cell culture (SILAC) is a powerful technique for comparative quantitative proteomics, which has recently been applied to a number of different eukaryotic organisms. Inefficient incorporation of labelled amino acids in cell cultures of Arabidopsis thaliana has led to very limited use of SILAC in plant systems. We present a method allowing, for the first time, efficient labelling with stable isotope-containing arginine and lysine of whole Arabidopsis seedlings. To illustrate the utility of this method, we have combined the high labelling efficiency (>95%) with quantitative proteomics analyses of seedlings exposed to increased salt concentration. In plants treated for 7 days with 80 mM NaCl, a relatively mild salt stress, 215 proteins were identified whose expression levels changed significantly compared to untreated seedling controls. The 92 up-regulated proteins included proteins involved in abiotic stress responses and photosynthesis, while the 123 down-regulated proteins were enriched in proteins involved in reduction of oxidative stress and other stress responses, respectively. Efficient labelling of whole Arabidopsis seedlings by this modified SILAC method opens new opportunities to exploit the genetic resources of Arabidopsis and analyse the impact of mutations on quantitative protein dynamics in vivo.
Sample Processing Protocol
A Dionex Ultimate 3000 nanoHPLC system was used with 2 µg of peptides injected onto an Acclaim PepMap C18 nano-trap column (Dionex). After washing with 2% (vol/vol) acetonitrile 0.1% (vol/vol) formic acid peptides were resolved on a 150 mm × 75 µm Acclaim PepMap C18 reverse phase analytical column over a 200 min organic gradient with a flow rate of 300 nl min−1. The chromatography performed for these samples was as follows. The gradient commenced with 4 minutes of 95% buffer A (0.1% formic acid)/5% buffer B (80% acetonitrile, 0.08% formic acid), followed by a linear gradient to 40% buffer B over 128 minutes, then an increase to 98% buffer B for 20 minutes duration, and completed with a return to 5% buffer B at minute 152 for 30 minutes. Ions accepted for MS/MS were 2+ and greater. Dynamic exclusion was set to 45 seconds, and the inclusion mass width for precursor ions was 10 ppm. The allowed number of missed trypsin cleavages was set to 2. Peptides were ionized by nano-electrospray ionization at 1.2 kV using a fused silica emitter with an internal diameter of 5 µm (New Objective). Tandem mass spectrometry analysis was carried out on a LTQ-Velos Orbitrap mass spectrometer (Thermo Scientific) using data-dependent acquisition, measuring and sequencing the top 15 ions.
Data Processing Protocol
Raw files were processed, quantified and searched using MaxQuant version 188.8.131.52 and the Andromeda peptide search engine searching against the Uniprot Arabidopsis thaliana database (updated September 2012). The variable modifications were set as oxidation of methionine; acetylation of the protein N-terminus; deamidation of asparagine and glutamine, glutamine conversion to pyroglutamate; as well as the heavy proline products to determine if loss of heavy label was due to arginine conversion. Fixed modifications were set to carbamidomethylation of cysteines only. The MS tolerance was set to 7 ppm with the MS/MS tolerance set to 0.5 Da. The peptide and protein False Discovery Rate (FDR) were both set to 1%, and the proteins used for quantitation and further analysis had 2 or more peptides assigned to them. Significance of fold changes was calculated using a one sample Benjamini-Hochberg t-test analysis with a 0.05 threshold value.
Lewandowska D, ten Have S, Hodge K, Tillemans V, Lamond AI, Brown JW. Plant SILAC: stable-isotope labelling with amino acids of arabidopsis seedlings for quantitative proteomics. PLoS One. 2013 Aug 20;8(8):e72207. eCollection 2013 PubMed: 23977254