The proteolytic 18O labeling method determines the relative ratios of individual proteins between two samples. This technique utilizes a protease and water (H216O and H218O) to produce labeled peptides; peptides in one sample incorporate 16O by labeling in H216O, and the other sample incorporates 18O by labeling in H218O. Both samples are mixed in a 1:1 ratio and subjected to mass spectrometric analysis to identify and quantify the proteins from which the peptides originated. Technical issues in sample preparation and data processing prevented this method from becoming widely accepted in the field of quantitative proteomics, however these problems have been resolved and the technique is now rapidly gaining popularity. This review focuses on the recent technological developments that have improved the reliability and practicality of proteolytic 18O labeling, including improved peptide labeling techniques, techniques to prevent proteasecatalyzed 18O to 16O back exchange reaction, mass spectrometry platforms suitable for this technique, computational tools for the calculation of 16O/18O-peptide ratios, and a new strategy that allows to compare a large number of samples.
Keywords: 18O labeling, isotope labeling, mass spectrometry, proteomics, quantitative proteomics, protein quantification, Electrophoresis, SILAC, LC-MS/MS, Proteolytic Enzymes, Hydrolysis, Chromatography, Glycoslation
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