Age-dependent changes in protein phosphorylation have been noted for individual proteins, e.g., the crystallins (Ueda et al. , 2002) and various sarcoplasmic reticulum proteins (Xu and Narayanan, 1998). The sensitive analysis of phosphopeptides and/or phosphoproteins requires a separation of phosphorylated peptides or proteins from excess nonphosphorylated species. While few proteomic studies have focused on the characterization of the ''phosphoproteome'' of aging tissue, much research effort has been placed on method development for the analysis of the phosphoproteome (Mann et al., 2002; Conrads et al., 2002). Importantly, antibodies against phosphoserine, phosphothreonine and phosphotyrosine allow for the enrichment of phosphoproteins prior to chromatographic separation and mass spectrometric analysis of phosphopeptides, resulting in a reduction of sample complexity (Granborg et al., 2002; Pandey et al., 2002). During mass spectrometric analysis, phospho-peptides can be recognized through specific fragmentation processes, i.e., the loss of PO^ (79 Da) from all phosphoamino acids in the negative electrospray ionization (ESI) mode or the neutral losses of HPO3 (80 Da) or H3PO4 (98 Da) from phosphoserine and phos-phothreonine in the positive ESI mode (Schlosser et al., 2001). Moreover phosphopeptides can be monitored through the appearance of characteristic immonium ions from phosphotyrosine (Steen et al. , 2002) or chemically modified (into dimethylamine-containing sulfenic acids) phosphoserine and phosphothreonine (Steen and Mann, 2002a). The resolution of such immonium ions from other small peptide fragments is possible because the reporter ions contain a higher incidence of mass-deficient atoms (O, P, S), creating an inherent Mass-Deficient Mass Tag (MaDMaT) (Steen and Mann, 2002b). The chemical modification of phosphoserine and phosphothreonine can be achieved after ^-elimination at alkaline pH, generating dehydroalanine, which can be derivatized via Michael addition of appropriate nucleophiles. This chemistry enables biotinylation of original phosphoserine and phosphothreonine residues for affinity purification (Oda et al., 2001), selective immobilization on solid phases (Zhou et al., 2001), and introduction of a cleavage-site for a lysine-specific protease (Knight et al., 2003).
A further reduction of sample complexity can be achieved by metal-affinity chromatography (IMAC) of phosphopeptides on chromatographic supports containing trivalent metal ions such as Fe3+ or Ga3+ (Zarling et al., 2000; Riggs et al., 2001; Ficarro et al., 2002; Chen et al., 2002). Optimal resolution of phosphorylated from nonphosphorylated peptides requires chemical esterification of peptide carboxylate groups (Ficarro et al., 2002), and the selectivity of the method towards phosphopeptides can be improved when proteins are digested with endoproteinase Glu-C instead of trypsin, reducing the number of acidic residues per peptide (Seeley et al., 2005). For relative quantitation of phospho-peptides, Ga(III) IMAC has been combined with stable isotope labeling of peptides at the N-terminal amino group (Riggs et al., 2005).
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