Computational protocol: Validating Antibodies to the Cannabinoid CB2 Receptor

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[…] Raw data were processed through the Proteome Discoverer software (version 1.3, Thermo Scientific) and analyzed by both an in-house Mascot server (version 2.3; and the SEQUEST program (Thermo Scientific). Spectra were searched against a subset of the NCBInr amino acid sequence database containing all entries matching the taxon Rattus (78244 sequence entries; download April 2012) including corrected sequence entries for the CB2 receptor protein expressed by the rat CB2 cannabinoid receptor cell line (CHO-K1; Chan Test Corp.; Cleveland, OH). All NCBInr amino acid sequence entries for rat CB2 receptor protein are inconsistent with the sequence published by , which has been used for the CHO-K1 cell line (Genbank accession number AF176350). We found amino acid variations in either position 224 (T or A) or 227 (Q or L) in combination with the variable C-terminus of the two isoforms of CB2. We therefore integrated all nine permutations between position 224, 227 and the variable C-terminus into the database. Searches were set up for full tryptic peptides with a maximum of two missed cleavage sites and carboxyamidomethyl cysteine, deamidated asparagine and oxidized methionine as variable modifications. The mass tolerance thresholds were set to 10 ppm for precursor ions and 0.8 Da for fragment masses. The significance thresholds for high-confidence Mascot ion scores and SEQUEST Xcorr were calculated by the Perculator algorithm () allowing a false discovery rate (FDR) of < 1% (q<0.01). Low-confidence identifications were also checked manually for false-negative identification of the CB2 receptor protein. Peak intensities measured in the targeted approach were analyzed manually using the Xcalibur software (Thermo Scientific). […]

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