4800 plus maldi tof toftm analyzer

Molecular mass and amino acid sequence of the purified peptides were determined using a 4800 Plus MALDI TOF/TOF^TM Analyzer (Applied Biosystems, Beverly, MA, USA). During analysis, each sample was desorbed and ionized at 337 nm and operated in positive ion delayed extraction reflector mode. Spectra were recorded over mass/charge (m/z) range of 100–1500. Mass spectrometry/mass spectrometry (MS-MS) experiments were achieved through collision-induced dissociation while peptide sequencing was performed via manual calculation.

All the protein spots of interest were selected and excised manually. Sequencing-grade trypsin (Promega, USA) was added for digestion overnight at 37°C and the enzymatic hydrolysate was collected. ZipTip (Millipore, USA) desalination was performed.
The samples were mixed with alpha-cyano-4-hydroxycinnamic acid (HCCA) matrix as a 1 : 1 relationship. The MS and MS/MS data for protein identification were obtained through 4800 Plus MALDI TOF/TOFTM Analyzer (Applied Biosystems). Combined peptide mass fingerprinting and MS/MS queries were performed using the MASCOT search engine 2.2 (Matrix Science, Ltd) embedded into GPS-Explorer Software 3.6 (Applied Biosystems) on the National Center for Biotechnology Information database.

The dried samples were dissolved in 2 μl of 20% acetonitrile and treated with supersaturated a-cyano-4-hydroxycinnamic acid (CHCA) matrix solution. The solvent composed of 0.5% TFA and 50% ACN. All the samples were analyzed on 4800 Plus MALDI TOF/TOFTM Analyzer (Applied Biosystems, United States) at a scan range of 800–4,000 Da with UV light at 355 nm using Nd:YAG laser. The data were searched using the International Protein Index human protein database with Mascot search engine.

Protein digestion and MALDI-TOF/MS analyses were performed as follows. Each protein gel piece was destained with 100 mM NH₄HCO₃ in 30% (v/v) acetonitrile (ACN) for 2 h at 40 °C. The gel pieces were minced, lyophilized and digested in 25 mM NH₄HCO₃ with 10 ng sequencing-grade modified trypsin (Promega, Madison, WI, USA) at 37 °C overnight. After digestion, peptides were extracted by three washes with 0.1% trifluoroacetic acid (TFA) in 60% ACN. The peptides were desalted by ZipTipC-18 pipet tips (Millipore, Bedford, MA, USA) according to the product manual. Tryptic peptide samples were analyzed using a 4800 Plus MALDI TOF/TOFTM Analyzer (Applied Biosystems, Foster, CA, USA).

The purified laccase was further separated by Native-PAGE. After the electrophoresis, the gel was stained with citrate-phosphate buffer (100 mM, pH 4.0) containing 1.0 mM ABTS and the laccase band was collected for further study. MALDI-TOF/TOF-MS analysis used commercial service provided by Sangon Biotech on 4800 Plus MALDI TOF/TOFTM Analyzer (ABI, Foster City, CA, United States). Mass spectra were obtained in positive ions regime using reflectron. The program Mascot¹ was used for protein identification by “peptides fingerprints” and fragmentation spectra. The database NCBI was used for searching homology among proteins of all organisms and fungi with the accuracy mentioned taking into account possible methionine oxidation by atmospheric oxygen and possible modification of cysteine by acrylamide (Younes and Sayadi, 2011 (link); Zheng et al., 2017 (link)).

Significantly up- or down-regulated protein spots were excised from gels and destained for 2 h at room temperature using a freshly prepared wash solution consisting of 100% acetonitrile, 50 mM NH₄CHO₃ (50:50 v/v). Proteins were digested using a trypsin solution according to an established method (Xiong et al., 2011 (link)). Peptide mixtures were analyzed using the 4800 Plus MALDI TOF/TOF^TM Analyzer (ABI), which is a matrix-assisted laser desorption ionization time of flight (MALDI-TOF/TOF) mass spectrometer. Mass spectrometry was completed using an established method (Li et al., 2011 (link)). Proteins were identified using peak lists for searches against the NCBInr database with the Mascot search engine (http://www.matrixscience.com/). The search criteria consisted of the following: Enzyme, Trypsin; Variable modifications, Oxidation (M); Peptide tolerance, 200 ppm; MS/MS tolerance, 0.8 Da; Instrument, MALDI-TOF/TOF; and Carbamidomethyl (C) as a fixed modification for all alkylated samples. Blast2GO software was used for gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of identified proteins (Conesa et al., 2005 (link)).