Mascot 2.4 search engine

Mass spectrometry analysis on recombinant proteins was carried out essentially as described in ref. [30] (link) with minor modifications. For sample preparation, the oxidizing H₂O₂-treatment of gel bands was replaced by reduction with dithiothreitol for 45 min at 56°C and alkylation with iodoacetamide for 35 min at room temperature. LC-MS/MS raw data were acquired on a LTQ-Orbitrap (Velos) hybrid mass spectrometer (ThermoFisher) as in ref. [30] (link). Peak lists were generated with the Mascot Distiller version 2.4.3 software (Matrix Science) from the LC-MS/MS raw data. Using the Mascot 2.4 search engine (Matrix Science), we searched all MS/MS spectra against the target-decoy version of an updated compilation of the A. thaliana protein database provided by TAIR (nuclear, mitochondrial, and plastid genome; TAIR v10.0; December 14, 2010; 35,386 entries) and a home-made list of contaminants (260 entries). The set of allowed variable modifications were acetyl (N-termini), methionine oxidation and dioxidation, methyl (Lys, Arg), dimethyl (Lys, Arg), and trimethyl (Lys). In addition, carbamidomethyl cysteine was set as a fixed modification. Mascot search results were automatically filtered as described in ref. [30] (link) with the IRMa 1.30.4 version software. Spectra of interest were checked manually to confirm sequence and modifications.

Gel spots of interest were excised and cut into 1 mm³ cubes, de-stained by washing in acetonitrile, and subjected to reduction and alkylation before in-gel digestion with trypsin at 37°C using a ProGest Investigator in-gel digestion robot (Digilab, Marlborough, MA) and standard protocols [57 (link)]. Digested peptides were extracted with 10% formic acid, and applied (0.5 μL) to the MALDI target along with α-cyano-4-hydroxycinnamic acid matrix (0.5 μL, 10 mg/mL, 50:50 acetonitrile: 0.1% trifluoroacetic acid) and allowed to dry. MALDI-mass spectrometry data were acquired, using a 4800 MALDI TOF/TOF analyzer (ABSciex, Cheshire, UK) equipped with a Nd:YAG 355 nm laser and calibrated using a mixture of peptides. The most intense peptides were selected for mass spectrometry (MS)/MS analysis using GPS Explorer (ABSciex) to interface with the Mascot 2.4 search engine (Matrix Science) and the MS/MS data using Mascot 2.4 directly. Swiss-Prot (Dec 2012) or NCBInr (Aug 2013) databases were interrogated using Homo sapiens as species restriction. The data were searched with tolerances of 100 parts-per-million for the precursor ions and 0.5 Da for the fragment ions, trypsin as the cleavage enzyme, assuming up to one missed cleavage, carbamidomethyl modification of cysteines as a fixed modification and methionine oxidation selected as a variable modification.

For intact mass determination, protein samples (20–30 μl, 20–30 μM) were desalted through an XTerra MS C8 2.1 × 10 mm HPLC column (Waters, Milford, MA) and eluted with an increasing acetonitrile gradient (2% (v/v) to 98% (v/v) acetonitrile in aqueous 1%(v/v) formic acid) and delivered into an electrospray ionization MS (LCT, Micromass, Manchester, UK) previously calibrated with a myoglobin standard. Multiply charged signals and their time of flight were obtained and deconvoluted using MaxEnt1 software.
For protein identification, peptide mass fingerprinting of tryptic in‐gel digests was performed using a 4800 MALDI TOF/TOF analyzer (Applied Biosystems, Foster City, CA). Protein/peptides (0.5 μl) were mixed with 0.5 μl alpha‐cyano‐4‐hydroxycinnamic acid matrix (10 mg/ml in 50:50 acetonitrile:0.1% trifluoroacetic acid) and left to dry on a MALDI plate. MALDI TOF/TOF data were analyzed using GPS Explorer (ABSciex, Warrington, UK) to interface with the Mascot 2.4 search engine (Matrix Science, London, UK).