Absolute Quantification of the G Protein-Coupled Receptor Rhodopsin by LC/MS/MS Using Proteolysis Product Peptides and Synthetic Peptide Standards

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Abstract
Methods for the absolute quantification of a membrane protein are described using isotopically labeled or unlabeled synthetic peptides as standards. Synthetic peptides are designed to mimic peptides that are cleaved from target analyte proteins by proteolytic or chemical digestion, and the peptides selected serve as standards for quantification by LC/MS/MS on a triple quadrupole mass spectrometer. The technique is complementary to relative quantification techniques in widespread use by providing absolute quantitation of selected targets with greater sensitivity, dynamic range, and precision. Proteins that are found to be of interest by global proteome searches can be selected as targets for quantitation by the present method. This method has a much shorter analytical cycle time (minutes versus hours for the global proteome experiments), making it well suited for high-throughput environments. The present approach using synthetic peptides as standards, in conjunction with proteolytic or chemical cleavage of target proteins, allows mass spectrometry to be used as a highly selective detector for providing absolute quantification of proteins for which no standards are available. We demonstrate that quantification is simple and reliable for the integral membrane protein rhodopsin with reasonable recoveries for replicate experiments using low-micromolar solutions of rhodopsin from rod outer segments.