M

Measure the peptide abundance ratio (L/H) based on the isotopic label

Search the database for peptide identification based on the isotopic label

FIGURE 32.2. (Continued)

Goshe et al. [31, 32] developed a novel strategy to identify and quantify the extent of protein phosphorylation using a phosphoprotein isotope-coded affinity tag (PhIAT). In this method (Figure 32.2B, C), Cys-residues are blocked and the phosphate groups of pSer and pThr residues are removed by hydroxide ion-mediated ^-elimination to produce thiolate-reactive sites. When comparing the relative phosphorylation states of phosphopeptides from two distinct samples, these thiolate-reactive sites are modified with isotopic versions of 1,2-ethanedithiol (EDT) that contain either four alkyl hydrogens (d0-EDT) or four alkyl deuteriums (d4-EDT). Once isotopically labeled, the d0/d4-EDT-labeled residues are biotinylated using (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine. The phosphorylated peptides are purified and concentrated using affinity chromatography and quantified by LC-MS/MS analysis. The relative stability of the PhIAT-label during CID enables the specific location of the site of phosphory-lation to be identified from the product ion spectrum and permits the state of phos-phorylation to be quantified [32]. An improved solid-phase-based version of PhIAT, termed phosphoprotein isotope-coded solid-phase tag (PhIST) [33], was developed in which isotope-coded solid-phase reagents containing either 12C6/14N or 13C6/15N and a UV-sensitive photolinker were used to increase efficiency and sensitivity for isolating and analyzing phosphopeptides. Because of its smaller tag and increased stability during CID, the PhIST labeling approach is more effective at identifying multiple phosphorylation sites on the same peptide while permitting the relative quantification of phosphorylation than PhIAT. Depending on the reaction conditions, the labeling of some protein samples can be substoichiometric, and difficulties associated with the low solubility of the EDT compound in aqueous solutions can promote protein precipitation. Recently, Goshe and colleagues [34] have addressed these issues by improving their PhIST approach to yield effective quantitative blocking of cysteinyl residues while promoting quantitative labeling of both pSer and pThr residues at the peptide level using Ba2+-catalyzed ^-elimination and Michael addition of (^,,R)-DTT as the thiolate linker, circumventing the difficulties encountered with labeling at the protein level. Current work is underway to optimize these labeling strategies for identification and quantification of LRR RLK phosphorylation sites in planta.

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