Protein extraction and mass spectrometric identification of methylated proteins in plant tissues

A mass of 250 mg of fresh E. grandis leaf or root tissues was harvested and snap frozen in liquid nitrogen. Tissues were then ground immediately after freezing in a sintered glass tissue grinder in either ice cold 1x IP Lysis/Wash Buffer (Thermo Scientific) supplemented with 1 mM plant protease inhibitor cocktail (Sigma Aldrich; Cat#P9599) for immunoprecipitation or in 50 mM Tris HCl (pH 8)/1% SDS/50 μM DTT/1% PVPP/1 mM plant protease inhibitor cocktail for total protein extraction and Western blotting. Grinding was performed on ice for no more than 3 min after which the soluble protein was quantified using the Qubit total protein analysis kit according to manufacturer’s instructions (Life Technologies). Protein extract was then diluted to 1 μg/μL using the extraction buffer and either used immediately for immunoprecipitation or was mixed with NuPAGE LDS buffer (Life Technologies) and snap frozen in liquid nitrogen for Western blotting.

Immunoprecipitation was performed using the Pierce IP immunoprecipitation kit (Thermo-Scientific; Cat#26148) where ASYM24 (Merck-Millipore; Cat#07-414) or anti-β-tubulin (Abcam; Cat#ab6046) was cross-linked to the agarose resin. Approximately 900 μg of total protein was added to the agarose slurry and incubated at room temperature for 2 h. Following incubation, rinses and elution of the bound proteins were performed according to manufacturer’s instructions.

For protein separation by electrophoresis, equal amounts of total protein were separated on a 4–20% Mini-PROTEAN® TGX™ gradient gel for 2 h at 80 V. The gel was stained overnight, either with Brilliant Blue G- Colloidal stain or SYPRO Ruby, at room temperature with shaking. Coomassie stained gels were destained in milli-Q water for 5 h. Protein bands were excised and further destained for 10 min in a 1:1 solution of 25 mM ammonium bicarbonate and acetonitrile. This was repeated until bands appeared colourless, and was followed by incubation with acetonitrile for 20 min. Bands in SYPRO Ruby stained gels were visualised by UV Transilluminator, bands were excised and then destained as per the Coomassie stained bands. All further treatments were identical for gel bands stained with either staining method.

The gel bands were incubated with 10 mM DTT in milli-Q water for 1 h at 37 °C to reduce cysteine residues, then with 25 mM IAA in milli-Q water for 1 h at 37 °C to alkylate the cysteine residues. Proteins were digested by the addition of Trypsin Gold (Promega) with a final enzyme concentration of 5 μg/mL and incubated overnight at 37 °C. While trypsin was used to cleave proteins, it has been reported to produce inconsistent [46] or ineffective cleavage at di-methylated arginine sites [56]. The digestion solution was collected into new low-binding tubes and peptides were further extracted by adding a 1:1 solution of acetonitrile and 0.1% formic acid and sonicated for 10 min. This process was repeated twice. Peptides were dried under vacuum (Waters) and were then resuspended in 0.1% formic acid.

LC-MS/MS analysis was performed on a Xevo QToF mass spectrometer from Waters (Micromass,UK) fed by a nanoAquity UPLC (Waters Corp., Milford, MA, USA) at the Western Sydney University mass spectrometry facility. 3 μl of digested peptides were loaded onto a nanoAquity UPLC Symmetry C18 trapping column (1.7 μm, 180 μm × 20 mm) and then separated and eluted from the column using a binary gradient program at a flow rate of 5 μl/min and desalted at this flow rate for 3 min. The peptides were washed off the trap at 400 nL/min on to a Waters C18 BEH analytical column (75 μm x 100 mm), packed with 1.7 μm particles with 130 Å pore size. After separation, the peptides were analysed using tandem mass spectrometry, implementing an emitter tip that tapers to 10 μm at 2300 V. Mobile phase A was 0.1% formic acid in water; and mobile phase B was 0.1% formic acid in acetonitrile. The nano-UPLC gradient was as follows: 0 min, 97:3 A/B; 5 min, 97:3 A/B; 75 min, 40:60 A/B; 85 min, 10:90 A/B; 97 min, 97:3 A/B; 110 min, 97:3 A/B. The mass spectrometer was operated in positive ESI mode with capillary voltage of 3.5 KV, cone voltage of 40 V, source temperature of 80 °C. Targeted MS/MS data or DDA (data dependent acquisition) data were acquired by continuously scanning for peptides of charge state 2⁺ to 4⁺ with an intensity of more than 50 counts per second; with a maximum of three ions in any given 3 s scan. Selected peptides were fragmented and the product ion fragment masses were measured. The data were acquired by the software Masslynx (Version 4.1, Micromass, UK).

The acquired DDA data from Masslynx with “RAW” extension, were converted to PKL files by Proteinlynx Global Server (PLGS) for analysis using the Mascot Daemon database (Australian Proteomics Computational Facility, Melbourne, Australia).

The MS/MS data files were searched against MSPnr100 database with trypsin as the enzyme. The following parameters were considered in the Mascot Editor tab for identification of the peptides: maximum missed cleavage of 3, peptide charge state of 2⁺ and 3⁺, peptide mass tolerance of 0.5 Da in MS and MS/MS data base, fixed modification: carbamidomethyl (C) and variable modifications: oxidation (M), mono and di-methylation of arginine (R). An ion score of 20 was applied to all results in order to filter out low probability matches. Peptides were also matched using Peaks Studio Software (version 7.5; Bioinformatics Solutions Inc., Waterloo, Ontario, Canada) by uploading the E. grandis protein list translated from the primary transcripts [24] and performing a database search to identify proteins and post-translational modifications. The following parameter settings were used: fixed modifications: carbamidomethyl (C); variable modifications: oxidation (M), mono and di-methylation of arginine (R); enzyme: trypsin; number of allowed missed cleavages: 3; peptide mass tolerance; 100 ppm; MS/MS mass tolerance: 0.5 Da; and peptide charge state: 2⁺ and 3⁺. Proteins identified as uncharacterised proteins were matched to their function by performing a BLAST search (Uniprot) and homology of the identified residues was checked using the alignment tool (Uniprot).

A recent report by Hart-Smith et al. [57] warned of false positive detection of methylated peptides when using ethanol, methanol and/or isopropanol in the preparation of large-scale mass spectrometry. The methods used here were different to the ones described by Hart-Smith [57] and the results were validated in the absence of alcohol-based staining. Furthermore, bacterially expressed tubulin β (which lacks methylation, Sigma; Cat#SRP5148) analysed from stained or unstained protocols, did not yield any falsely identified methylated arginine residues using the mass spectrometry protocol described above.