Strategies Relying on Chemical Derivatization

Overall there are two different kinds of derivatization techniques, which are commonly used for the relative quantification of phosphorylation. The first is based on j elimination and Michael addition. In this approach, two samples (proteins or peptides) to be compared are derivatized with differently deuterated forms (i.e., d0/d5) or different isotopes (i.e., C12/C13) of the same nucleophile, the samples are mixed, proteins are digested, and peptides are subjected to chromatography, which is directly coupled to a mass spectrometer. The ratio of the corresponding peptide pairs leads to a relative quantification of phosphorylation. In the second derivatiza-tion strategy, peptides derived from two different samples (control and treated) are derivatized with the undeuterated and the deuterated form of methanol, thus converting carboxyl residues into their corresponding methyl esters in the process of methylesterification. Phosphopeptides are then usually enriched to overcome the low level of phosphorylation and peptide pairs are analyzed by MS as described above. The major drawbacks of these approaches are the side reactions observed in chemical derivatization and elution differences of the peptide pairs in chromatography, depending on the type of derivatization agent. Especially deuterated forms of the peptides are known to exhibit a slight retention time shift in chromatography when compared to their nondeuterated counterparts, and this sometimes impairs direct comparison of peptide pairs [99]. Also, the number of samples that can be compared is limited due to the limited availability of differentially deuterated or isotopically coded tags.

Other strategies involving stable isotope labeling by metabolic labeling or tagging (like SILAC or iTRAQ) are not primarily applied to phosphoproteins/peptides and are not further discussed [for reviews see references 97 and 100].

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