Zheng et al. BMC Genomics 2011, 12:40 http://www.biomedcentral.com/1471-2164/12/40 10 mM NH4H2PO4 in 0.1% TFA (15 seconds) to pro- vide matrix (a-cyano-4-hydroxycinnamic acid, HCCA) thin layer preparations for MALDI TOF/TOF analysis using an Ultraflex III spectrometer (Bruker, Germany). MS measurements were performed in positive ion reflector mode with 20 kV accelerating voltage and 23 kV reflecting voltage, and spectra were calibrated using PeptideCalibStandard II (Bruker, Germany) of mass range 800-3200 Da as external standards [47]. WARP-LC 1.0 software controlled the precursor ion selection and trig- gered the MASCOT (Matrix Science) database search, and BioTools 3.0 was used for data visualization and post pro- cessing. All these software packages were provided by Bru- ker Daltonics GmbH, Germany. SDS PAGE and MALDI-TOF/TOF MS measurements Nine bands corresponding to complexes with high stain- ing intensity were excised from BN gel and incubated in 1% (w/v) SDS/1% (v/v) b-mercaptoethanol solution for 1 h. After a rapid rinse with water nine bands were loaded on top of a 5% stacking and 12% separating gel. The gap between the bands and the gel was filled with 1% low melt agarose. Electrophoresis was performed at 4°C, starting for 3 h at 80 V, and the voltage was then raised to 150 V overnight (max. 50 mA). Spots were excised from the Coomassie blue-stained gel and sub- jected to in-gel digestion protocol. The digestion mix- ture was dissolved in 2 μl 0.1% TFA prior to MALDI- TOF MS analysis. One microliter of the analyte solution, along with equivalent matrix solution (HCCA), was mixed and applied onto 600 μm AnchorChip target for MALDI-TOF/TOF analysis. MS measurements were performed as described above. Data Interpretation and Database Searching The raw MS/MS data were searched against a compo- site target/decoy protein sequence database using the program MASCOT 2.1 http://www.matrixscience.com. The target component comprised 3,952 protein sequences derived from M. bovis BCG Pasteur 1173P2 (downloaded from the National Centre for Biotechnol- ogy Information database (NC_008769)) and protein sequences of known contaminant proteins, including trypsin and human keratin. Moreover, the decoy compo- nent was composed of the reversed sequences of all pro- teins in the target component. Search parameters allowed for up to two missed tryptic cleavage sites, the carbamidomethylation of cysteine, the possible oxidation of methionine, and monoisotopic mass type. Further- more, peptide mass tolerance was ±0.2 Da and MS/MS tolerance was ±0.6 Da. For positive identification, the Mowse score of the result of (-10 × Log (P)) had to be over the significance threshold level (P < 0.05). More- over, identification of individual proteins matched with Page 9 of 11 two or more unique peptides was considered as valid identification. Bioinformatics analysis The physicochemical characteristics of all identified pro- teins were analyzed by some software. The theoretical Mr, pI value and average GRAVY score were obtained from the Swiss-Prot and TrEMBL databases http://us. expasy.org/tools/protparam.html. Predictions of trans- membrane topology and possible signal sequence in the proteins were conducted using the TMHMM 2.0 pro- gram and SignalP program, respectively, both of which were publicly available from the Centre for Biological Sequence Analysis at the Technical University of Den- mark http://www.cbs.dtu.dk/services. Functional associa- tion prediction between identified proteins was analyzed using STRING version 8.3 interaction database http:// string.embl.de/. Functional classifications of the identi- fied proteins were determined using the Pasteur Insti- tute functional classification tree http://genolist.pasteur. fr/BCGList/. Additional material Additional file 1: List of the identified proteins by MS analysis showing the sequence of all the identified peptides by independent MS/MS fragmentation. The charge state of each peptide is +1. Accession: the identifier number from the National Centre for Biotechnology Information database; Protein ID: protein name in M. bovis BCG database; Score: the Mascot score of the protein identification; MW [kDa]: molecular weight in kDa of the respective protein; pI: isoelectric point of the protein, calculated from its amino acid sequence; SC[%]: sequence coverage in percent, calculated from the identified peptides of the respective protein; RMS [ppm]: the RMS value of the deltas between the calculated and experimental masses of the peptides, which belong to a particular protein; Inten1.: sum of all intensity values related to 100 laser shots for all peptides, which belong to a particular protein; S/N1: sum of all S/N-values for all peptides, which belong to a particular protein; MH+ (calc) [Da]: calculated mass of the singly protonated peptide; Δm [Da]: difference between the measured and calculated masses of the respective peptide in Dalton; Inten2: the sum of the MS peak intensities per 100 laser shots; S/N2: S/N ratio of the respective peptide; Sequence: the sequence of the corresponding peptide. Abbreviations ACN: acetonitrile; AG: arabinogalactan; BCG: bacillus Calmette-Guérin; BN: blue native; CBB: Coomassie Brilliant Blue; DDM: n-dodecyl-β-D-maltoside; 2- DE: two-dimensional gel electrophoresis; EMB: ethambutol; FadD: fatty acid degradation; GRAVY: grand average of hydrophobicity; Hsp: heat shock protein; IEF: isoelectric focusing; IMP: integral membrane protein; kbp: kilobase pair(s); kDa: kilodalton; LC-MS/MS: liquid chromatography tandem mass spectrometry; MALDI TOF: matrix-assisted laser desorption/ionization time of flight; Mr: molecular mass; PAGE: polyacrylamide gel electrophoresis; PDIM: phthiocerol dimycocerosate; pI: isoelectric point; PKS: polyketide synthase; PPS: phenolpthiocerol synthase; RT: rome temperature; SDH: succinate dehydrogenase; SDS: sodium dodecyl sulphate; SL: sulfolipid; TB: tuberculosis; TFA: trifluoroacetic acid; TMH: transmembrane helix Acknowledgements We are grateful to Professor Zongde Zhang(Beijing Tuberculosis and Thoracic Tumor Research Institute, P.R. China)for providing strain M. bovis var