A more detailed version of the methods is available as “Supplementary data”. Primary WT mouse thoracic (T1–T4) DRG suspensions were prepared as described above and plated onto specialized cell culture dishes (Sarstedt, Nümbrecht, Germany). Membrane and soluble fractions from approximately 80,000 cells per experimental group (control or LPS; 4 separate platings) were obtained using the ProteoExtract Native Membrane Protein Extraction Kit (EMD Millipore, MA, USA) and processed as per the manufacturer’s instructions and stored at − 80 °C. Protein concentrations were determined by NanoDrop BioTek ELx808 (BioTek Instruments Inc., VT, USA). Protein samples were reduced, alkylated, and digested with trypsin in-solution following a modified version of a previously published protocol49. Tryptic peptides were first subjected to strong cation exchange (SCX) fractionation using a SCX SpinTips sample preparation kit (Protea Biosciences, Morgantown, WV, USA), according to manufacturer’s instructions, before they were analyzed by liquid chromatography-tandem mass spectrometry (LC–MS/MS). MS analysis was performed using an Agilent 6550 iFunnel quadrupole time-of-flight (QTOF) mass spectrometer equipped with an Agilent 1260 series LC instrument and an Agilent Chip Cube LC–MS interface (Agilent Technologies, Mississauga ON, Canada). Chromatographic separation of peptides was accomplished using a high-capacity Agilent HPLC Polaris Chip and a linear gradient solvent system consisting of formic acid, water, and acetonitrile. Positive-ion electrospray tandem mass spectral data were collected over a mass range of 100–1700 mass/charge.

Tandem mass spectra were extracted from raw data and processed against the mouse NCBI non-redundant database and a custom database (containing all known mouse RAGE protein isoforms) using Spectrum Mill (Agilent Technologies Canada Ltd., Mississauga, ON, CA). Search parameters included a fragment mass error of 50 parts per million (ppm), a parent mass error of 20 ppm, trypsin cleavage specificity, and carbamidomethyl as a fixed modification of cysteine. Variable modifications included: carbamylated and acetyl-lysine, oxidized methionine, pyroglutamic acid, deamidated asparagine, and phosphorylated serine, threonine, and tyrosine. Spectra were also searched against semi-trypsin non-specific C- and N-termini. Spectrum Mill results were validated at peptide and protein levels (1% false discovery rate, FDR), and spectral counts and intensities were used to report relative quantification of proteins. Mass Profiler Professional (MPP, version 15.0, Agilent, Santa Clara, CA, USA) software was used for statistical analysis using one-way ANOVA. A cut-off value of p < 0.05 and the Benjamini and Hochberg FDR set at < 1% were used to obtain statistically significant results. In addition, a fold change (FC) of ≥ 2 and < 0.5 in spectral intensities with respect to control were considered to classify proteins as up- and down-regulated, respectively.

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