Mascot software v 2

Colloidal coomassie blue G-250 staining was used to visualize CyDye-labeled protein features in 2-DE followed by excised interested post-stained gel pieces for MALDI-TOF MS identification. The detailed procedures for protein staining, in-gel digestion, MALDI-TOF MS analysis and the algorithm used for data processing were described in our previous publication [17 (link)]. The spectrometer was calibrated with a peptide calibration standard (Bruker Daltonics), and internal calibration was performed using trypsin autolysis peaks at m/z 842.51 and m/z 2211.10. Peaks in the mass range of m/z 800–3000 were used to generate a peptide mass fingerprint that was searched against the Swiss-Prot/TrEMBL database (released on November 2011) with 533 049 entries using Mascot software v2.3.02 (Matrix Science, London, UK). The following parameters were used for the search: Homo sapiens; tryptic digest with a maximum of 1 missed cleavage; carbamidomethylation of cysteine, partial protein N-terminal acetylation, partial methionine oxidation and partial modification of glutamine to pyroglutamate and a mass tolerance of 50 ppm. Identification was accepted based on significant MASCOT Mowse scores (p < 0.05), matched peptide sequence coverage higher than 15%, spectrum annotation and observed versus expected molecular weight and pI on 2-DE.

Spots of interest were manually excised and processed automatically in a Proteineer DP (Bruker Daltonics, Bremen, Germany). The sample was digested and prepared as previously described^{58 (link)}, with minor modifications^{59 (link)}. For MALDI-TOF/TOF analysis, samples were automatically acquired in an ABi 4800 MALDI TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA, USA) in positive ion reflector mode (ion acceleration voltage, 25 kV for MS acquisition and 1 kV for MS/MS). PMF and MS/MS fragment ion spectra were smoothed and corrected to zero baseline using routines embedded in ABi 4000 Series Explorer Software v3.6. Each PMF spectrum was internally calibrated with the mass signals of trypsin autolysis ions to reach a typical mass measurement accuracy of <25 ppm. GPS Explorer v4.9 was used to submit the combined PMF and MS/MS data to MASCOT software v.2.1 (Matrix Science, London, UK), searching in the non-redundant NCBI protein database. The mascot total score is −10*Log(P), where P is the probability that the observed match is a random event. The percentage of sequence coverage was calculated as the ratio of amino acids (number identified in peptides / number of theoretical peptides from sequence data).

For MALDI-TOF/TOF analysis, samples were automatically acquired in an ABi 4800 MALDI-TOF/TOF mass spectrometer (Applied Biosystems) in positive ion reflector mode (the ion acceleration voltage was 25 kV to MS acquisition and 1 kV to MSMS) and the obtained spectra were stored into the ABi 4000 Series Explorer Spot Set Manager. PMF and MSMS fragment ion spectra were smoothed and corrected to zero baseline using routines embedded in ABi 4000 Series Explorer software v. 3.6. Each PMF spectrum was internally calibrated with the mass signals of trypsin autolysis ions to reach a typical mass measurement accuracy of less than 25 ppm. Known trypsin and keratin mass signals as well as potential sodium and potassium adducts (+21 and +39 Da) were removed from the peak list. To submit the combined PMF and MS/MS data to Mascot software v. 2.1 (Matrix Science, London, UK), GPS Explorer v. 4.9 was used, searching in the non-redundant NCBI protein database. The mass tolerance for precursors was set to ±50 ppm and to ±0.3 Da for MS/MS fragment ions. Peptide identifications were accepted when scored at greater than 95.0% probability by the Mascot algorithm [23 (link)].

For MALDI-TOF/TOF analysis, samples were automatically acquired in an ABi 4800 MALDI TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA) in positive ion reflector mode (the ion acceleration voltage was 25 kV for MS acquisition and 1 kV for MS/MS) and the obtained spectra were stored into the ABi 4000 Series Explorer Spot Set Manager. Peptide mass fingerprinting (PMF) and mass spectrometry/mass spectrometry (MS/MS) fragment ion spectra were smoothed and corrected to zero baseline using routines embedded in ABi 4000 Series Explorer Software v3.6. Each PMF spectrum was internally calibrated with the mass signals of trypsin autolysis ions to reach a typical mass measurement accuracy of <25 ppm. Known trypsin and keratin mass signals, as well as potential sodium and potassium adducts (+21 Da and +39 Da, resp.), were removed from the peak list. To submit the combined PMF and MS/MS data to MASCOT software v.2.1 (Matrix Science, London, UK), GPS Explorer v4.9 was used, searching in the nonredundant NCBI protein database.

Cells were detached from culture plates by scraping in PBS containing EDTA, washed twice with ice-cold PBS, resuspended in a buffer containing 65 mM DTE, 65 mM CHAPS, 9 M urea, 35 mM Tris-base, and disrupted by sonication in an ice bath. 60 μg of proteins/sample were submitted to 2-DE as previously described [40 (link)]. Digitalized images were obtained by ImageScanner III and then qualitatively and quantitatively analyzed by the ImageMaster software (GE Healthcare BioSciences, Piscataway, NJ, USA). The increasing/decreasing index (fold change) was calculated as the ratio of spot relative volume between the different gel maps. Protein spot identification was obtained by MALDI-ToF/MS as previously described [41 (link)]. Mascot software v.2.2 (Matrix Science, Boston, MA, USA) was used to identify spots from the NCBI non-redundant database. Candidates with a score > 81 (corresponding to p ≥ 0.05 for a significant identification) were further evaluated by comparison with experimental coordinates from 2-DE.