Chromatography conditions on NanoAcquity The analysis was performed using a nanoACQUITY Ultra- Performance-LC (UPLC; Waters). The samples were trapped on a 2060.18 mm, 5 mm Symmetry C18 precolumn (Waters), and the peptides were separated on a ACQUITY UPLCH BEH130 C18 column (Waters), 75 mm6200 mm, 1.7 mm particle size. The solvent system consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Trapping was performed during 3 min at 5 mL/min with 99% of solvent A and 1% of solvent B. Elution was performed at a flow rate of 400 nL/ min, using 1–40% gradient (solvent B) over 35 min at 45uC followed by 65% (solvent B) over 5 min. MS and MS/MS conditions on SYNAPT mass spectrometer The MS and MS/MS analyzes were performed using a SYNAPTTM apparatus, an hybrid quadrupole orthogonal accel- eration time-of-flight (TOF) tandem mass spectrometer (Waters) equipped with a Z-spray ion source and a lock mass system. The capillary voltage was set at 3.5 KV and the cone voltage at 35 V. Mass calibration of the TOF was achieved using phosphoric acid (H3PO4) on the [50;2000] m/z range. Online correction of this calibration was performed with Glu-fibrino-peptide B as the lock- mass. The ion (M+2H)2+ at m/z 785.8426 was used to calibrate MS data and the fragment ion (M+H)+ at m/z 684.3469 was used to calibrate MS/MS data during the analysis. For tandem MS experiments, the system was operated with automatic switching between MS and MS/MS modes (MS 0.5 s/ scan on m/z range [250;1500] and MS/MS 0.7 s/scan on m/z range [50;2000]). The three most abundant peptides (intensity threshold 60 counts/s), preferably doubly and triply charged ions, were selected on each MS spectrum for further isolation and CID fragmentation with 2 energies set using collision energy profile. Fragmentation was performed using argon as the collision gas. The complete system was fully controlled by MassLynx 4.1 (SCN 566; Waters). Data analysis and protein identification Raw data collected during nanoLC-MS/MS analyses were processed and converted with ProteinLynx Browser 2.3 (Waters) into .pkl peak list format. Normal background subtraction type was used for both MS and MS/MS with 5% threshold and polynomial correction of order 5, and deisotoping was performed. The MS/MS data were analyzed using the MASCOT 2. 2. 0. algorithm (Matrix Science) to search against the UniProtKB/ Swiss-Prot database, version 54.8, concatenated with reversed copies of all sequences (26349,480 entries). Spectra were searched with a mass tolerance of 15 ppm for MS and 0.07 Da for MS/MS data, allowing a maximum of one missed cleavage site by trypsin and with carbamidomethylation of cysteine residues and oxidation of methionine residues specified as fixed and variable modifica- tions, respectively. Protein identifications were validated when at least two peptides were found with Mascot ion score greater than 35 for each MS/MS spectrum. For the estimation of the false positive rate in protein identification, a target-decoy database search was performed. Criteria used for protein identifications followed the general guidelines for reporting proteomic experiments (MIAPE; http:// www.psidev.info). Surface plasmon resonance analysis BIAcore 3000 system (Biacore AB) was used to evaluate the binding of P140 peptide to HSC70 protein. Sensor chip CM5, P140 Peptide in Lupus surfactant P20, amine coupling kit containing N-hydroxysuccini- mide (NHS) and N-ethyl-N9-dimethylaminopropyl carbodiimide (EDC), 2-(2-pyridinyldithio)ethaneamine (PDEA) and ethanol- amine were from Biacore AB. Biosensor assays were performed with HBS-EP buffer as running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20, pH 7.4). The different compounds were diluted in the running buffer. The sensor chip surface was regenerated after each experiment by injecting 10 mL of 10 mM HCl. Recombinant bovine HSC70 (Stressgen) was immobilized on flow cells of a CM5 sensor chip through its thiol groups using 35 mL PDEA in 50 mM borate buffer, pH 8.3 on the NHS/EDC-activated matrix. Then, 35 mL of HSC70 (100 mg/mL in formate buffer, pH 4.3) were injected until a response of 13,000 response units (RU) corresponding to 13 ng/mm2 of HSC70 was immobilized. Twenty mL of a 50 mM cysteine/1 M NaCl solution was used to saturate unoccupied sites on the chip. The direct binding measurement of P140 peptides to HSC70 was carried out at 25uC with a constant flow rate of 20 mL/min. P140 peptide and analogues were injected in the flux at different concentrations for 3 min, followed by a dissociation phase of 3 min. The kinetic parameters were calculated using the BIAeval 3.1 software on a personal computer. Analysis was performed using the simple 1:1 Langmuir binding model. The specific binding profiles were obtained after subtracting the response signal from the control empty channel and from blank- buffer injection. The fitting to each model was judged by the x square value and randomness of residue distribution compared to the theoretical model. Immunofluorescence To study the co-localization of HSC70 and P140 peptide staining, freshly isolated splenocytes from MRL/lpr mice were washed with PBS and incubated at RT for 45 min in PBS containing 25 mM biotinylated P140. Cells were then washed three times in Tris buffered-saline (TBS) and fixed for 1 h at RT in 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB). Fixed cells were incubated for 1h at RT with a rat monoclonal anti- HSC70 antibody labeled with R-phycoerythrin (PE; 1:150; clone 1B5; Stressgen) in TBS containing 2% (w/v) BSA. After washing in TBS, cells were incubated with fluorescein isothiocyanate (FITC)-labeled streptavidin (1:400, Molecular Probes) and 49,69- diamidino-2-phenylindole (DAPI, 1:1,000; Molecular Probes), washed thrice in TBS and fixed for additional 1 h at RT in 4% PFA in 0.1M PB. Control experiments were performed by omitting biotinylated peptide. Samples were finally mounted on slides using Dako’s fluorescence mounting medium. Pictures were obtained with a Zeiss fluorescence microscope (Axiovert 200 M) using an X63 magnification. Images were acquired with a digital camera using Axiovision 4.37 software (Zeiss). Double immuno- fluorescence images were obtained by superimposing two single- color pictures with Adobe Photoshop 8.0. To study the localization of HSC70 in permeabilized cells, see the legend of the Figure S2. Immuno-electron microscopy To study the subcellular localization of HSC70 by immunoelec- tron microscopy, freshly isolated PBLs from MRL/lpr mice were washed with PBS and fixed in 4% (v/v) PFA and 0.2% (v/v) glutaraldehyde in 0.1M PB for 1h at RT. Cells were embedded in 1% agarose, permeabilized with 0.2% saponin for 10 min, washed and saturated with 2% BSA and 0.2% (w/v) water fish skin gelatin (WFSG, Sigma) in TBS before overnight incubation at 4uC with anti- HSC70 rat monoclonal antibody (1:200; clone 1B5; Abcam) in TBS containing 0.2% BSA and 0.05% WFSG. Bound antibody was visualized by preembedding labeling using goat anti-rat IgG PLoS ONE | www.plosone.org 10 April 2009 | Volume 4 | Issue 4 | e5273