Jh. Till et al., USE OF SYNTHETIC PEPTIDE LIBRARIES AND PHOSPHOPEPTIDE-SELECTIVE MASS-SPECTROMETRY TO PROBE PROTEIN-KINASE SUBSTRATE-SPECIFICITY, The Journal of biological chemistry, 269(10), 1994, pp. 7423-7428
To search for peptides which serve as substrates for protein kinases,
an approach based on peptide libraries has been developed. These pepti
de libraries are chemically synthesized by a modified ''divide-couple-
recombine'' strategy. After reaction with the kinase of interest, the
most highly phosphorylated substrate (selected from the library) is id
entified using on-line liquid chromatography-electrospray mass spectro
metry (LC-ESMS). Negative ion LC-ESMS with stepped collision energy is
used to identify phosphorylated peptides in the enzyme reactions. As
predicted, the cAMP-dependent protein kinase is shown to preferentiall
y phosphorylate Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) in a library co
nsisting of 19 variants of Kemptide substituted at position 2. Additio
nal experiments have been carried out on the nonreceptor tyrosine kina
se v-Abl using a peptide library based on the v-Src autophosphorylatio
n site -Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly). These result
s indicate that Ile is the optimal residue at the position N-terminal
to tyrosine. Individual peptides containing the Glu-Asp-Ala-Ile-Tyr mo
tif have V-max/K-m values 6-fold higher than the peptide based on the
autophosphorylation site itself, confirming the results of the library
experiments. This motif has been identified in several tyrosine kinas
es at a position in the sequence not previously reported to serve as a
phosphorylation or autophosphorylation site.