Tandem Affinity Purification and Liquid Chromatography–Mass Spectrometry/Mass Spectrometry Analysis

Plant materials (30 g) of either cell suspensions harvested 7 days after subculturing (suspension experiments #1, #2, and #3 representing three biological replicates) or 12-day old seedlings (seedlings experiment #1) overexpressing TaMAB2-TAP recombinant protein (line EM4), were homogenized in liquid nitrogen. The same procedure was applied for SerRS-TAP (Kekez et al., 2016) used as a control sample. Crude protein extracts were prepared in an equal volume (w/v) of extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) Nonidet P-40, 1 mM DTT, 1 mM PMSF, 1x Roche complete protein inhibitor), at 4°C. The soluble protein fraction was obtained by centrifugation at 4°C. The extract was passed through a 0.45-µm filter. Purifications were performed as described by Rigaut et al. (1999), with some modifications. Briefly, total protein extract was incubated for 3 h at 4°C under gentle rotation with 300 µl of IgG-Sepharose 6 Fast Flow beads (GE Healthcare) pre-equilibrated with 10 ml of extraction buffer. IgG-Sepharose beads were transferred to a 1-ml Mobicol column (MoBiTec) and washed with 30 ml of immunoglobulin G (IgG) wash buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% (v/v) glycerol, 0.1% Nonidet P-40, 1 mM DTT), and 10 ml of tobacco (N. tabacum L.) etch virus (TEV) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% (v/v) Nonidet P-40, 0.25 mM EDTA, 1 mM DTT). Bound complexes were eluted via AcTEV digest (50 units; Invitrogen) overnight at 4°C followed by wash with 500 µl of TEV buffer, and then 3 ml calmodulin binding buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% (v/v) Nonidet P-40, 10 mM β-mercaptoethanol, 1 mM imidazole, 2 mM CaCl₂, 1 mM magnesium acetate). The CaCl₂ concentration of the IgG-eluted fraction was adjusted to 2 mM, and the fraction incubated for 1 h at 4°C under gentle rotation with 200 µl of calmodulin-agarose beads (Stratagene) pre-equilibrated with 10 ml of calmodulin binding buffer. Calmodulin-agarose beads were washed with 30 ml of calmodulin binding buffer and packed into a Mobicol column. Bound complexes were eluted with 2.5 ml of calmodulin elution buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% (v/v) Nonidet P-40, 10 mM β-mercaptoethanol, 1 mM imidazole, 22 mM EGTA) and precipitated with trichloroacetic acid (20%, v/v). The protein pellet was washed with ice-cold acetone, redissolved in sample buffer, and separated on 4–12% gradient gel (Roth). Proteins were visualized with colloidal Coomassie Brilliant Blue staining. Parts of gels with proteins were cut into small (1 mm²) slices and further processed for MS analysis. Gel particles were washed three times with 5 mM ammonium bicarbonate (ABC) and 50% acetonitrile, dehydrated with 100% acetonitrile, reduced with 10 mM DTT in 20 mM ABC at 56°C, alkylated with 55 mM iodoacetamide and 20 mM ABC in the dark. After a two-step washing procedure with 5 mM ABC and 50% acetonitrile gel, slices were dehydrated in 100% acetonitrile and rehydrated in 50 µl of digest buffer containing 625 ng of trypsin (MS Gold; Promega). After 10 min 150 µl 20 mM ABC was added and proteins were digested at 37°C overnight. The resulting peptides were extracted with three serial washing steps: the first in 30% acetonitrile, 3% trifluoroacetic acid, the second in 80% acetonitrile and 0.5% acetic acid, and the third in 100% acetonitrile. Acetonitrile was evaporated using a vacuum centrifuge. Peptides were concentrated and purified with StageTips as described by Rappsilber et al. (2007). Briefly, StageTips were wetted by methanol and equilibrated by solution A (0.5% CH₃COOH). Peptides were acidified by solution A, and loaded onto equilibrated tips. Salts were washed by solution A, and peptides were eluted from tips by solution B. Anhydrous acetonitrile was evaporated from desalted and purified peptides by vacuum centrifugation. Peptides were separated and then measured by a nanoscale HPLC system Easy-nLC (Proxeon Thermo) coupled to an LTQ-Orbitrap Discovery mass spectrometer (Thermo Scientific) through a nano-electrospray ionization source (Proxeon Thermo). Briefly, peptides were loaded onto C18 nanocolumn, made in-house by slurry packing PicoFrit capillaries (New Objective, 75 µm fused silica diameter, 10 µm tip diameter) with Luna 3 µm C18(2) material (Phenomenex). Peptides were eluted by a linear gradient of solution B in solution A (from 3 to 35% B in 45 min), and electrosprayed directly into the mass spectrometer. The mass spectrometer measured peptides in an orbitrap analyzer (one million ions at 30,000 resolution setting). In parallel, top 20 peptides were fragmented in a linear ion trap (3,000 ions) using dynamic exclusion to prevent recurring fragmentation of prominent peptides.

Raw data was processed by MaxQuant software as described by Cox and Mann (2008). UniProt Arabidopsis complete proteome set was searched using Andromeda (Cox et al., 2011) or X!Tandem (Craig and Beavis, 2004). UniProt ID numbers were obtained from mass spectrometry-based protein identification and searched against databases of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/protein) and the Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org/) to annotate each gene. Target proteins were selected according to three criteria: firstly, they were presented by at least two peptides of which at least one was unique. Secondly, they were not presented in control experiment in which seryl-transfer ribonucleic acid (seryl-tRNA) synthetase was used as bait (proteins were considered as seryl-tRNA synthetase partners if they were presented by at least two peptides of which at least one was unique in any of the SerRS experiments). Thirdly, they were found in at least two independent experiments.