DOI: 10.6084/m9.figshare.22903031
title: .Raw files from Thermo Scientific Orbitrap Fusion Lumos Tribrid Mass Spectrometer

description: Immature mo-DCs were seeded in T-25 cell culture flasks at a density of 2-15 x 106 cells in 7.5 mL of RPMI 1640 and 10% fetal bovine serum, supplemented with 2 mM L-glutamine and 20 µM ß-mercaptoethanol. Immature mo-DCs were stimulated with 1 µg/mL purified LTA or live <em>B. burgdorferi</em> strains at a multiplicity of infection of 10 bacteria per cell for 24 hours. Cells were incubated at 37°C, 5% CO2, and &gt;95% humidity. At the end of the incubation period, cells were harvested and pelleted at 1200 RPM for 10 minutes at 4oC. Culture flasks were washed with cold PE buffer, adherent cells were lifted, and this solution was used to pellet cells. Cells were resuspended in 400 µL/2 x 106 cells in lysis buffer (1% CHAPS, pepstatin, leupeptin, chymostatin, antipain, PMSF and EDTA) and lysed for 1 hour at 4oC in a rocking table. Cellular debris was removed by centrifugation at 10,000 RCF for 15 minutes to clear supernatant twice. Twenty µg of the anti-HLA-DR antibody L243 (purified from ATCC HB-55 hybridoma) was added to each sample and incubated overnight at 4oC. One hundred µL of Rec-Protein G-Sepharose 4B conjugate (Thermo Fisher Scientific) slurry was added to the antigen-antibody complex and incubated with gentle mixing for 2 hours at room temperature. The agarose-antibody-antigen complex was washed with 500 µL of a 20mM Tris, pH 7.4 and 150mM NaCl solution. Peptides were eluted from the bead-antibody complex in 100 µL of 1% trifluoroacetic acid (TFA) and isolated using C18 spin columns (Thermo Fisher Scientific) according to manufacturer’s instructions. Samples were buffer exchanged in Waters Oasis MAX (Mixed-mode Anion eXchange) 96-well microelution plates using a Waters Positive Pressure-96 Processor prior to mass spectrometry analysis. Each Oasis well was conditioned with 100 µL of 4% H3PO4, samples were diluted 1:1 with 4% H3PO4, and transferred to the microelution plate. Pressure was applied to concentrate the peptides on the MAX phase, washed twice with 50 µl of 5% NH4OH followed by two 50 µL washes of 20% acetonitrile. The flow through was discarded and a new clean 96-well plate was placed under the Oasis MAX plate. Peptides were eluted from the MAX phase with two 50 µL aliquots of 75% acetonitrile containing 1% TFA. Eluted peptides were dried down by speed vacuum centrifugation and reconstituted in 2% acetonitrile, 0.1% formic acid. Peptides were injected into a C18 trap column and separated by a 2% to 90% acetonitrile gradient containing 0.1 % formic acid at 300 nL per minute over 90 minutes on a 75 um x 50 cm ReproSil-Pur C18 AQ 3 µm in-house packed New Objective PicoFrit column using a Thermo Fisher Easy-nanoLC1000 system interfaced with a Thermo Scientific Orbitrap Fusion Lumos Tribrid Mass Spectrometer. Peptides were analysed with a data dependent 3 second cycle fragmentation method for the highest abundant precursors. Survey and MS2 scans were performed at 120,000 and 30,000 resolutions, respectively. The mass spectrometry *.raw files were searched with the PEAKS X software against the <em>B. burgdorferi</em> RefSeq database of protein sequences with no enzyme designation and allowing for variable modification of methionine oxidation and asparagine or glutamine deamidation. A stringent threshold for peptide identification in the PEAKS X software was set to -10lgP values ≥20 (~p-value≤ 0.01) to filter the identified peptides at a 1% false discovery rate using the PEAKS decoy-fusion algorithm. Using the PEAKS software we compiled a list of all peptides from all subjects (Table I) and a list of all peptide clusters based on proteins of origin (Table II).