Download Peptide microarrays for detailed, high-throughput

Application Note - Serine Threonine Kinases; Mechanistic Study
Peptide microarrays for detailed, high-throughput substrate identification,
kinetic characterization, and inhibition studies on protein kinase A
Riet Hilhorst, Liesbeth Houkes, Adriënne van den Berg, Rob Ruijtenbeek; PamGene International B.V. the Netherlands
Study Design
A PamChip® containing 140 serine/threonine peptides
was used for real-time detection of protein kinase A (PKA)
for substrate identification and kinetic characterization of
PKA (figure 1). Figure 2 shows a time course of peptide
phosphorylation over 50 minutes.
In this assay the IC50 of three PKA inhibitors, AMP–PNP,
staurosporin, and PKA inhibitor peptide, was investigated
as a function of ATP conc. (0.5 – 4.0 times Km) and
CREB peptide conc. (0.22 -3.3 times Km) (table 1).
Key Findings
In comparison to singleplex kinase activity assays, data
quality was high, variation in assay conditions and
reagent consumption were reduced considerably.
PKA was shown to phosphorylate many peptides
containing known PKA phosphorylation sites as well as
some new substrates.
Data in table 1 shows that staurosporine is a full ATP
competitive inhibitor whereas AMP-PNP has a different
inhibition mechanism.
“Author Quote”
The kinetic readout gives high-quality data; the
simultaneous incubation eliminates differences in medium
composition and experimental conditions.
Figure 1: A) Fluorescent image of a PamChip® peptide microarray
comprising 140 peptide phosphorylation substrates after 30 minutes of
incubation with complete PKA assay mix. B) Image of an identical array
after 30 minutes without PKA. On some peptides, low background binding
of the antibody is observed.
Figure 2: Time course of PKA-catalyzed phosphorylation on the array for a
number of peptides representing known protein phosphorylation
substrates. The peptide ID is based on the UniProt Knowledgebase, and
the numbers indicate the position of the first and last amino acids of the
peptide in the complete protein (UniProt annotation and numbering).
Table 1: -logIC50 for PKA and the inhibitors AMP–PNP, staurosporin, and
PKA inhibitor peptide at different peptide and ATP concentrations.
Background
As a first step toward understanding the behavior of
kinase inhibitors in a cellular context, and ultimately in
vivo, the effect on the target kinase is usually studied in
vitro. Using standard kinase assays, such an approach is
laborious and time-consuming, whereas the use of a
peptide microarray makes such investigations feasible in
a short time frame while using very small amounts of
reagents such as recombinant expressed kinases.
References:
Hilhorst R. et al., Anal Biochem. 2009 Apr 15;387(2):150-61.
Conclusion
PamChip® STK peptide arrays allow profiling of PKA and are used in potency determination of PKA inhibitors as function of
peptide substrate. STK arrays allow identification of peptide substrates for STK kinases that are still uncharacterized.
©2013 PamGene International B.V.
All rights reserved
Application note: #201007 (9-2010)
Contact us:
Wolvenhoek 10, 5211 HH Den Bosch, the Netherlands
+31 73 615 80 80,  [email protected]