18O Stable Isotope Labeling. Differential 16O/18O coding relies on the 18O exchange that takes place at the Cterminal carboxyl group of proteolytic fragments, where two 16O atoms are typically replaced by two 18O atoms by enzyme-catalyzed oxygen exchange in the presence of H218O [109] . The resulting mass shift between differentially labeled peptide ions permits identification, characterization, and quantitation of proteins from which the peptides are proteolytically generated. In contrast to ICAT, 18O labeling does not favor peptides containing certain amino acids (e.g., cysteine), nor does it require an additional affinity step to enrich for these peptides [110] . Unlike iTRAQ, 16O/18O labeling does not require a specific MS platform nor does it depend on fragmentation spectra (MS2) for quantitative peptide measurements. It is amenable to the labeling of human specimens (e.g., plasma, serum, tissues), which represents a limitation of metabolic labeling approaches (e.g., SILAC). Taken together, recent advancements in the homogeneity of 18O incorporation, improvements made on algorithms employed for calculating 16O/18O ratios and the inherent simplicity of this technique should result in increased use of 18O labeling [111] . In general, 18O labeling suffers from two potential drawbacks, inhomogeneous 18O incorporation and inability to compare multiple samples within a single experiment. A dual 18O labeling using a non-gel-based platform has been developed to overcome the major problems of existing proteolytic 18O labeling methods [112] .