Covalent tagging of phosphorylated peptides by phosphate-specific deoxyribozymes.

Many natural peptides and proteins are phosphorylated on tyrosine (Tyr) or serine (Ser) residues. Phosphorylated peptides are important within neurochemistry (neuropeptides), immunology (cytokines), and endocrinology (hormones). For such peptides as well as for larger proteins, side chain phosphorylation is frequently associated with modulation of biological function.[1] Methods for analysis of phosphopeptides often depend upon their initial chromatographic separation from nonphosphorylated analogues using support-bound chelators or covalent binders of phosphate groups or products derived from them.[2] Alternatively, phosphotyrosine-specific antibodies can be generated, albeit with the attendant investments in cost and time.[2g,3] Here we describe proof of principle for an entirely different approach to phosphopeptide analysis in which DNA catalysts (deoxyribozymes) covalently tag phosphorylated amino acid side chains of peptides. In this approach, it is critical to ensure high selectivity for modification of phosphorylated amino acid side chains over their nonphosphorylated analogues. Deoxyribozymes were originally identified for the catalysis of RNA cleavage,[7] and their use has expanded to encompass a range of chemical reactions.[8] Our lab has reported a variety of deoxyribozymes for different chemical reactions,[9] including the covalent modification of amino acid side chains.[5,10] In particular, we have recently shown that tripeptide substrates can be covalently modified by the attachment of an RNA strand at nonphosphorylated Tyr or Ser.[6] Here we sought to identify deoxyribozymes that covalently modify phosphorylated TyrP (YP), using in vitro selection as shown in Figure 1. The hexapeptide substrate AAAYPAA was connected to a DNA anchor oligonucleotide via either a short or long tether [see structures in Supporting Information; the short tether connects the hexapeptide directly via its α-amino group to the DNA anchor, whereas the long tether includes an intervening hexa(ethylene glycol) moiety]. In vitro selection was used to identify deoxyribozymes that attach a 5′-triphosphorylated RNA tag to TyrP, with pyrophosphate as the leaving group. Figure 1 Strategy for selective covalent modification of phosphorylated Tyr (TyrP, YP) within a peptide substrate. In vitro selection identifies deoxyribozymes that function with a TyrP-containing hexapeptide substrate, catalyzing attachment of 5′-triphosphorylated ... Two new deoxyribozymes from the selection process, 8VM1 and 8VP1 (one from each of the two selection experiments), were examined in more detail on the basis of their high catalytic activities with the TyrP- and analogous phosphoserine (SerP)-containing hexapeptides. Both deoxyribozymes were highly selective (>200:1) for each phosphorylated peptide over its nonphosphorylated analogue, with no detectable reaction at TyrOH or SerOH (<0.5%; Figure 2). 8VM1, which was identified by selection with the short tether, favored as its substrate the TyrP peptide over the SerP peptide by about 4- to 5-fold. In contrast, 8VP1, which was found via selection with the long tether, functioned equally well with TyrP or SerP peptides. 8VP1 was also found to accept a range of different amino acid identities—including hydrophobic and charged residues—flanking the TyrP that it covalently modifies (Figure 3), suggesting broad generality for different phosphopeptide sequences. Figure 2 The 8VM1 and 8VP1 deoxyribozymes covalently modify phosphotyrosine and phosphoserine. (a) PAGE image showing high selectivity for TyrP over TyrOH and for SerP over SerOH (50 mM HEPES, pH 7.5, 40 mM Mg2+, 20 mM Mn2+, 150 mM NaCl, 37 °C; single-turnover ... Figure 3 Sequence generality for the phosphopeptide substrate. Covalent modification by 8VP1 was examined with DNA-anchored hexapeptide substrate CAAYPAA and several illustrated sequence variants, for which one amino acid adjacent to YP (on either side) was changed ... To examine the applicability of deoxyribozymes for analysis of mixtures of phosphorylated and nonphosphorylated peptides, such a mixture (each peptide attached via a disulfide to the DNA anchor) was tagged with RNA by the 8VP1 deoxyribozyme. Analysis of the unpurified product mixture by MALDI mass spectrometry (after DTT cleavage of the DNA anchor) revealed selective RNA tagging of only the phosphopeptides, despite the presence of a large amount of nonphosphorylated peptide (Figure 4). Figure 4 Analysis of a peptide mixture by mass spectrometry using the DNA-catalyzed tagging approach. See diagram of this experiment in the Supporting Information. Each of a mixture of nonphosphorylated and phosphorylated peptides (100 pmol nonphosphorylated peptides; ... In summary, we have demonstrated proof of principle that DNA can catalyze highly selective covalent modification of phosphorylated Tyr or Ser residues in phosphopeptides by attaching an RNA tag at those positions. To our knowledge, this is the first report of any chemical approach for covalent, specific tagging of phosphopeptide side chains. In downstream applications, this RNA tag should be useful to report upon the amount of phosphorylated peptides present in a sample, e.g., by RT-PCR, which may help to avoid issues encountered during mass spectrometric analysis of peptide phosphorylation.[11] A wide range of peptide sequence contexts are accepted by the investigated deoxyribozymes, suggesting that this general approach may be made competitive with more traditional chromatographic separations of phosphopeptides.[2] The phosphopeptide analytical approach outlined here is distinct from methods that depend upon engineering of individual kinases to accept modified ATP substrates.[12] The present findings also expand the repertoire of DNA catalysis to include covalent modification of phosphorylated amino acid side chains. Independently, we have shown that RNA-tagging deoxyribozymes can discriminate against phosphorylated residues in favor of their nonphosphorylated analogues with promising selectivity (>20:1; data not shown). That observation along with the present work suggests the viability of ratiometric analyses in which both phosphorylated and nonphosphorylated peptides are covalently modified with different tags in the same sample. Several important issues must be addressed in future development of this approach. We will seek DNA catalysts that tag specific sequences of phosphopeptides, rather than accepting a broad range of peptide sequences. The approach also must be developed to work with free, rather than oligonucleotide-anchored, peptide substrates as well as with large phosphorylated proteins, ideally in complex mixtures such as cell lysates. Towards this goal, we have recently demonstrated the first steps towards DNA-catalyzed reactivity of free peptides;[6,13] such efforts must be merged with the present work to establish a useful analytical method.