These rules are commonly used to distinguish between aliphatic and aromatic phosphoamino acids. A by electrospray ionization-tandem mass spectrometry. Our experiments clearly demonstrate hydroxylation and subsequent phosphorylation of a proline residue in -crystallin A in the eye as well as in heart tissue of rat. Keywords:Hydroxyproline, Mass Spectrometry (MS), Peptide Conformation, Post-translational Modification, Protein Phosphorylation, Hydroxyamino Acids, Hydroxylation, Phosphoamino Acids, Phosphoprotein == Introduction == A wide range of biological processes are controlled by reversible protein phosphorylation, and conservative estimates indicate reversible phosphorylation targets of up to one-third of cellular proteins (1). To understand protein regulation through phosphorylation it is essential to find and characterize all sites and types of phosphorylation on proteins and to identify the kinases involved. Besides the three genetically encoded hydroxyamino acids serine (Ser), threonine (Thr), and tyrosine (Tyr), there are two other hydroxyamino acids, hydroxyproline (Hyp)2and hydroxylysine (Hyl). They are not encoded in the genome, although they can be identified in mature proteins. They are generated by enzymatic hydroxylation of proline (Pro) and lysine (Lys) residues (2). All genetically encoded hydroxyamino acids in polypeptide chains, such as serine, threonine, and tyrosine have been shown that they can undergo post-translational phosphorylation (3,4). Although phosphorylation of Hyl has been described in collagens (5) phosphohydroxyproline (Hyp(P)) has not been identified in native proteins. In addition to theO-phosphorylation of the hydroxyl group,O-glycosylation is another important modification. There is a growing speculation that phosphorylation and glycosylation share a reciprocal relationship. Both Hyp and Hyl are known to undergo glycosylation in a few proteins. In collagens up to 70% of Hyl residues are modified with galactose and glycosyl-galactose (6). Hyl was also found to be AOH1160 phosphorylated in collagens (5). In extensins (hydroxyproline-rich glycoproteins) of the cell wall in plants, 50% of protein mass is glycosylated AOH1160 at Hyp residues by mono-, di-, tri-(12)–linked arabinoses (7,8,9). Furthermore, cytoplasmic F box-binding protein SKP1 inDictyosteliumcontains a pentasaccharide linked to Hyp (10). These findings suggest that subsequent modification of Hyp is widespread and that, in analogy to all other hydroxyamino acids, phosphorylation of Hyp should be possible in native proteins. Hydroxylation of Pro is the precondition for subsequent phosphorylation and has been studied extensively in collagens (2). The hydroxyl groups are introduced by sequence specific prolyl-3- or prolyl-4-hydroxylases. Prolyl-3-hydroxylase modifies Pro residues located C-terminally of glycine (Gly-Pro motif). Although Pro located at the N-terminal side of Gly (Pro-Gly motif) is hydroxylated by prolyl-4-hydroxylase, both hydroxylases introduce the hydroxyl group intransconfiguration. In collagens >90% of Hyp is intrans-4 configuration. It seems to be the most common Hyp isomer whereas only up to 2% aretrans-3-Hyp (2). The alternativecisconfiguration of Hyp was not described in native proteins so far. Although Hyp was found in a multiplicity of other proteins, the corresponding hydroxylases are mostly unknown. Several experiments have shown that enzymatic phosphorylation of Hyp residues in synthetic peptides by a AOH1160 Ser-specific kinase is possible, although the kinetics of Hyp phosphorylation were very low compared with those of Ser and Thr (1113). The configuration of Hyp played a crucial role in these experiments. Enzymatic phosphorylation oftrans-4-Hyp appeared to be possible, but not the phosphorylation ofcis-4-Hyp (12). All of these findings implicate that Hyp(P) is a proteinogenic amino acid. Here, we present the phosphorylation of Hyp in proteins by a specific polyclonal antibody. Furthermore we identified for the first time the sequence position of Hyp(P) by mass spectrometry in -crystallin A. Because the isomeric configuration of the Hyp residue remained unclear for the majority of Hyp-containing proteins, we outline the mass spectrometrical characterization of peptides containing various Hyp(P) isomers. Thus, it was interesting to know whether MS/MS fragmentation was able to characterize the configuration of Hyp(P) in post-translationally modified proteins and to find parameters that would allow for the identification of Hyp(P) in unknown proteins. This would permit to distinguish between Hyp(P) and other phosphoamino acids. == EXPERIMENTAL PROCEDURES == == == == == == Materials == Amino acid derivatives were purchased from Sigma-Aldrich and polystyrene AM RAM resin was obtained from Rapp Polymere (Tbingen, Germany). Peptide synthesis was performed with reagents and Rabbit Polyclonal to ZADH1 solvents from Applied Biosystems. All reagents and solvents for phosphorylation were obtained from Sigma-Aldrich or Merck and were of the highest available purity. == Protein Purification == Total protein was prepared from aorta, heart, and eye tissue of a female adult rat in consideration of the conservation ofO-phosphoesters.O-Phosphates are acid-stable and more or less unstable in alkali for example, whereasN-phosphates are acid-labile (14). Thus, it is important to prevent the phosphoamino acid residues from dephosphorylation by unsuitable chemical conditions or by phosphatases because in most cases only a small percentage of the potential phosphoprotein is phosphorylated at a specific point in time (15). The rat was maintained in the animal facility of the Heinrich-Heine-University, Dsseldorf, Germany. The tissues were frozen in liquid nitrogen and pulverized. Homogenization was performed with a.
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