4700 maldi tof tof proteomics analyzer

The two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique was performed as previously described47 (link). Total protein extracts, isoelectric focusing and SDS-PAGE gels were prepared as described elsewhere21 (link). Gels were silver stained through a mass spectrometry (MS) compatible protocol48 . PageRuler^TM (Fermentas) was used as size marker. In silico analysis of the 2D gels was performed in PDQuest 2-D (Bio-Rad). Spots with a significant statistical difference between strains (biological duplicates) were considered using the following criteria: P > 0.05 and two-fold change.
Protein spots were excised from gels and digested with trypsin. Samples were analysed using the 4700 Proteomics Analyzer MALDI-TOF/TOF (Applied Biosystems) as previously described49 (link). Data were analysed using GPS Explorer (Version 3.6; Applied Biosystems). Protein identification was performed combining data from PMF (Peptide Mass Fingerprint) and tandem mass (MS/MS) spectra. The Mascot (Matrix Science, UK) algorithm was used to determine the statistical significance (score > 52) of protein identification against the genome database of S. natalensis ATCC 27448.

Protein spots of interest were excised from 2-DE gels, washed, dried, and enzymatically digested with trypsin. The extracted peptide solution was subjected to the Applied Biosystems 4700 Proteomics Analyzer MALDI-TOF/TOF for MS/MS analysis. The peptide masses were searched against the National Center for Biotechnology Information (NBCI) database using Mascot Search from Matrix Science. A criterion for significance was defined using a probability-based molecular weight search (MOWSE) score. Individual scores greater than 56 indicated identity or extensive homology (P<0.05).

The protein in-gel digestion was accordding to Zhang et al. [19 (link)] with some changes. Protein bands were excised from the gels and washed with double-distilled water and then transferred to sterilized Eppendorf tubes. Then the protein bands were distained with 50 mM NH₄HCO₃ in 50% ethanol for 2 h at 40°C. The proteins therein were then reduced with 10 mM DTT in 50 mM NH₄HCO₃ and alkylated with 55 mM iodoacetamide in 50 mM NH₄HCO₃ for 1 h at room temperature. The proteins were digested overnight at 37°C by adding 15 μL of trypsin (Promega Co., Madison, WI, USA). The resulting peptides were extracted by washing the gel pieces with 0.1% trifluoroacetic acid in 67% ACN. Tryptic peptide masses were analyzed by a 4700 MALDI-TOF/TOF Proteomics Analyzer (Applied Biosystems, Carlsbad, CA, USA). Proteins were identified by searching against the NCBInr Malus domestica (apple) and Rocaseace database using an in-house MASCOT server v 2.1 (Matrix Science Co., London, UK).

The manually excised protein spots were digested with trypsin and then mixed with matrix solution for analysis using a 4700 MALDI-TOF/TOF Proteomics Analyzer (Applied Biosystems) according to the manufacturer’s instructions72 (link).

Liver proteins were labeled with the CyDye DIGE fluor minimal labeling kit (GE Healthcare, Piscataway, NJ). For isoelectric focusing electrophoresis, the labeled samples were placed in a strip holder using a step gradient protocol (30 v for 6 hrs, 60 v for 6 hrs, 200 v for 1 hr, 500 v for 1 hr, 1000 v for 1 hr and 8000 v for 6 hrs). After equilibrated in SDS equilibration solution, proteins in the strips were separated on a 12.5% homogeneous SDS-PAGE gel. DIGE images were visualized with the Typhoon Trio variable imager (GE Healthcare) and analyzed using DeCyder software v6.5 (GE Healthcare, Piscataway, NJ). The ratios of the log-standardized protein spot abundances (differences in expression) between the groups were computed and analyzed with a one-way ANOVA. Differentially expressed protein spots were excised from the stained gel for identification with 4700 MALDI-TOF/TOF Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA).

To identify differentially expressed proteins, spots of interest were excised manually from Coomassie brilliant blue stained polyacrylamide gels and subjected to in-gel digestion as previously described^{43 (link)}. The excised proteins were analyzed using a 4700 MALDI-TOF/TOF proteomics analyzer (Applied Biosystems). A peptide mass fingerprinting search and a combined search (+MS/MS) were performed using GPS Explorer™ software (Applied Biosystems) in non-redundant NCBI database of proteins using MASCOT searching engine as described previously^{44 (link)}.

Protein spots were manually excised from preparative CBB-stained gels and digested with sequencing-grade modified trypsin (Promega, Madison, WI, USA) following the manufacturer's protocol, then incubated at 37°C for 12 h. Tryptic peptides were redissolved in 0.8 µl of matrix solution [α-cyano-4-hydroxycinnamic acid (Sigma, St. Louis, MO, USA) in 0.1% trifluoroacetic acid, 50% acetonitrile] before application to the MALDI plate.
After air dying, samples were analyzed using a 4700 MALDI-TOF-TOF Proteomics Analyzer (Applied Bio-systems, Foster City, CA, USA). Combined MS and MS/MS spectra were submitted to MASCOT (V2.1, Matrix Science, London, UK) by GPS Explorer software (V3.6, Applied Biosystems). Database searches were used the following parameters: NCBInr database (release date: 2010.07.01); taxonomy of green plant; trypsin digest with one missing cleavage; no fixed modifications; MS tolerance of 100 ppm; MS/MS tolerance of 0.6 Da; and possible oxidation of methionine. MASCOT protein scores (based on combined MS and MS/MS spectra) greater than 75 were considered statistically significant (P≤0.05). The individual MS/MS spectrum with the most statistically significant (confidence interval of 95%) ion score (based on MS/MS spectra) was accepted.