Secreted Proteins of Leishmania Alter Macrophage Figure 6. Modulation of translocation of transcription factors AP-1 and NF-kB following exoproteome incubation. B10R macrophages were untreated (Nil), infected with L. mexicana or incubated with exoproteome for 16 h and stimulated with 100 ng/ml of LPS for 1 hour the following day. (A) AP-1 is degraded after infection and exoproteome incubation. No increase in translocation in response to LPS can be seen. (B). A noncanonical form of NF-kB translocates to the nucleus following LPS-stimulation of infected macrophages (p35/p50). Induction of this form of NF- kB can also be seen after stimulation of the exoproteome-treated macrophages. Furthermore, induction of the normal p65/p50 in response to LPS is greatly reduced compared to Nil. S, specific competition (100-fold excess of specific nonradioactive oligonucleotide incubated with nuclear proteins of Nil+LPS); NS, nonspecific competition (100-fold excess of nonspecific, nonradioactive SP-1 oligonucleotide incubated with nuclear proteins of Nil+LPS). doi:10.1371/journal.pone.0018724.g006 on a Biobasic C18 Integrafrit (New Objective) capillary column, using a Nano high-performance liquid chromatography system (1100 series unit; Agilent). Eluted peptides from the in solution digestion were electrosprayed as they exited the column and were analyzed on a QTRAP 4000 linear ion trap mass spectrometer (SCIEX/ABI) whereas peptides from the in gel digestion were analyzed on a QTof micro (Waters Micromass) mass spectrometer. Protein database searching Individual sample tandem mass spectrometry spectra were peak listed using Distiller version 2.1.0.0 (http://www.matrixscience. com/distiller.html) software with peak picking parameters set at 1 as for Signal Noise Ratio (SNR) and at 0.3 for Correlation Threshold (CT) for QTRAP 4000 data and at 5 SRN and 0.4 CT for micro QToF data. The peak-listed data was then searched against a copy of the NCBI GenBank database by using Mascot (Matrix Science, London, UK; version 2.1.4.04). Mascot was set up to search the Leishmania (Taxonomy ID: 5658) database (release 10th October 2008; 25,140 sequences protein entries) assuming the digestion enzyme trypsin, with a fragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 1.5 Da. Iodoacetamide derivative of cysteine was specified in both search engines as a fixed modification. Oxidation of methionine residues was specified in Mascot as a variable modification. Scaffold (version Scaffold_2_05_02, Proteome Software Inc) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm [35]. Protein identifications were accepted if they could be established at greater than 90.0% probability and contained at least 1 identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm [35]. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Bioinformatic analyses Gene ontology (GO) annotations were attributed using Blast2GO [36]. The initial Blastp step was performed against NCBI nonredundant database with E-value of 161023 and high scoring segment pair cut off 33. 50 top blast hits were retrieved and used for annotation. Blast2GO default parameters were used for the annotation step; the pre-eValue-Hit-Filter was 161026, annotation cut-off was 55 and GO Weight was 5. Annotation was augmented by using the Annotation Expander (ANNEX) and further by addition of the GO terms associated with functional domains derived from scanning the InterPro database [37,38]. Signal peptide prediction was performed using SignalP 3.0 [39]. PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e18724