Protein kinase substrate profiling with a high-density peptide microarray

Xiaoming Han, Tatsuhiko Sonoda, Takeshi Mori, Go Yamanouchi, Takayuki Yamaji, Syuhei Shigaki, Takuro Niidome, Yoshiki Katayama

    Research output: Contribution to journalArticlepeer-review

    15 Citations (Scopus)


    We describe a powerful peptide microarray for profiling protein kinase substrates that combines the merits of chemoselective immobilization of peptides to achieve high density spots with the advantages of fluorescence-based analysis of phosphorylation for nonhazardous detection. For detection of on-chip phosphorylation, we used a fluorescence-labeled antiphosphotyrosine antibody to detect phosphotyrosine and a biotinylated Phostag, which was subsequently bound with a fluorescence-labeled streptavidin for phosphoserine/threonine. More than 290 kinds of Tyr peptides and over 1,100 kinds of Ser/Thr peptides were chemoselectively immobilized onto a glass surface in a highdensity format to profile a panel of protein kinases, including c-Src, c-Abl, EGFR, JNK1, ERK2, p38α, and PKA. Many novel, highly reactive and specific peptides were identified as substrates for each protein kinase. Most substrates had the consensus motifs that have been reported previously but some new motifs were also found. The identification of two designed peptides that have higher reactivity than the famous PKA substrate (Kemptide) indicates that analysis of the amino acid biases of substrates is very helpful to the design of new substrates with high reactivity. Thus, the high-density peptide microarray is expected to be a powerful approach for high-throughput discovery of potential substrates for protein kinases.

    Original languageEnglish
    Pages (from-to)777-789
    Number of pages13
    JournalCombinatorial Chemistry and High Throughput Screening
    Issue number9
    Publication statusPublished - Nov 2010

    All Science Journal Classification (ASJC) codes

    • Drug Discovery
    • Computer Science Applications
    • Organic Chemistry


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