ProteinLynx software (Version 2.2.5), provided by the manufacturers, was used to analyze raw MS and MS/MS spectra and to Dibutyryl-cAMP ic50 generate a peak list which was introduced in the in-house Mascot MS/MS ion search software (Version 2.2, Matrix Science, Boston, MA) for protein identification.
NCBI was used as sequence database. Search parameters were as follows: LY2874455 molecular weight fixed modifications carbamidomethyl (C), variable modifications pyro-Glu (N-term Q) and oxidation (M), peptide tolerance 30 ppm, MS/MS tolerance 0.3 Da, charge state +2 and +3, enzyme trypsin, allowing up to 1 missed cleavage. Data analysis MS data were subjected to gene ontology analysis with Blast2GO, using default parameters [57]. Identified proteins were divided into classes for functional and localization analysis; data produced by the software were used for generation of graphs by Microsoft Excel. Acknowledgements We thank Prof. Christine Citti for kindly providing the type strain PG2T, Dr. Mario Ferrer-Navarro for his helpful suggestions during optimization of the NVP-BGJ398 clinical trial protein fractionation approach, Dr. Vittorio Tedde and Dr. Alessandro Tanca for assistance during electrophoresis and MALDI-MS identification, and Dr. Stefania Ghisaura from Biosistema Scarl for the DIGE experiments. This work was supported by funding
from the Grant “”Ricerca Sanitaria Finalizzata, Anno 2007, UPB S02.04.010, Cap SC02.1106″”, and Misura P5 Biodiversità animale (Regione Sardegna). Electronic supplementary material Additional file 1: 2-D PAGE map of liposoluble proteins from M. agalactiae PG2 T illustrating the protein identifications obtained by MS on the 3-10NL pI Interval. (DOC 175 KB) Additional file 2: 2-D PAGE map of liposoluble proteins from M. agalactiae PG2 T illustrating
the protein identifications obtained by MS on the 7-11 pI Interval. (DOC 166 KB) Additional file 3: 2-D PAGE map of liposoluble proteins from M. agalactiae PG2 T illustrating the protein identifications obtained by MS on the 4-7 pI Interval. (DOC 94 KB) Additional file 4: Table listing all protein identifications obtained from 2-D PAGE maps. The proteins listed in this table were identified from 2-D PAGE maps of the M. agalactiae PG2T Triton X-114 Epothilone B (EPO906, Patupilone) fraction. Maps are represented in Additional files 1 (pH 3-10NL), 2 (pH 7-11) and 3 (pH 4-7). (DOC 568 KB) Additional file 5: Protein profile of liposoluble proteins before and after precipitation. Right: approach used for GeLC-MS/MS characterization. The bars indicate the regions cut from the PAGE gel and subjected to mass spectrometry characterization. Protein identifications are reported in additional file 6, from top to bottom. (DOC 96 KB) Additional file 6: Table listing all protein identifications obtained by GeLC-MS/MS of the M. agalactiae PG2 T Triton X-114 liposoluble fraction. The protein profile used and the number of slices are reported in Additional file 5.