Feb

23

Peptide Arrays – Protocol

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11. Antimicrobial Peptide Arrays for Detection of Inactivated Biothreat Agents By: Chris R. Taitt1 , Stella H. North1, Nadezhda V. Kulagina1

Arrays of immobilized antimicrobial peptides are used to detect bacterial, viral, and rickettsial pathogens, including inactivated biothreat agents. These arrays differ from the many combinatorial peptide arrays described in the literature in that the peptides used here have naturally evolved to interact with and disrupt microbial membranes with high affinity but broad specificity. The interaction of these naturally occurring peptides with membranes of pathogens has been harnessed for the purpose of detection, with immobilized antimicrobial peptides acting as “capture” molecules in detection assays. Methods are presented for immobilizing the antimicrobial peptides in planar arrays, performing direct and sandwich assays, and detecting bound targets.

Affiliation(s): (1) US Naval Research Laboratory, Washington, DC, USA

Book Title: Peptide Microarrays: Methods and Protocols Series: Methods in Molecular Biology | Volume: 570 | Pub. Date: Aug-01-2009 | Page Range: 233-255 | DOI: 10.1007/978-1-60327-394-7_11

Subject: Protein Science

Key Words: Biothreat – detection, array – antimicrobial peptide

Nov

20

Application Note – Large Scale Mapping of Kinase Substrate Specificity

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Quantitative mapping of substrate specificity for protein kinase Src on a microarray
Chip Design

  • PKS – peptide microarray
  • 27 known protein tyrosine kinase substrates and their sequence variants
  • All peptides on the chip contain 8-10 redundancies.
  • Positive controls – synthetic phosphopeptides (pY phosphotyrosine incorporated by synthesis)
  • Negative controls – Ala substitution of Tyr at the phosphorylation site
  • Src kinase, p60c-src (Invitrogen)

Assay

  • Sample – Src kinase, p60c-src (Invitrogen)
  • Enzyme concentration – 0.5 mg/ml
  • Volume – 50 µl
  • Reaction time – 30 minutes or longer

Detection

  • Fluorescent dye is used to specifically stain phosphate groups (pS, pT, and pY).
  • Only the phosphate group of the phosphopeptides will be specifically stained by the fluorescent dye.  No need for antibodies which are known to suffer from non-specific or weak binding problems. If specially request, antibody detection can be applied.

Results

  • Figure 1 – Fluorescent image (inverted) of 4K kinase profiling microarray
  • (A) Image of a subset of four replicates of the I(Y/A)GEF, pY and Y sequences – The result shows that IYGEF is phosphorylated.
  • (B) Image of the YVPM (column 1) and the YEEIP (column 2) related sequences in double replicates – Among these, YEE and YEEI are phosphorylated by the Src kinase used, and YEEIP is a substrate of lower reactivitiy, and YVPM has the lowest reactivity.  Their results are consistent with the literature information.

Figure 1

kinase_app1_fig1

Figure 2 – Plot of the relative phosphorylation efficiency of each Y-containing peptide

  • The phosphorylating efficiencies of YEEI and IYGEF surpassed 90% of the synthetic pYEEI or pYGEF.
  • Except YLEL, Src kinase selected peptides with Glu, Asp or amino acids with small side chains (Gly or Ala) at the P+1 position. (ratio>30%, a line placed)
  • This was also demonstrated by Songyang’s paper (Songyang, Z and Cantley, L.C., (1995) Recognition and specificity in protein tyrosine kinase-mediated signaling, Trends in Biology Sciences, 20, 470-475).  [abstract]

kinase_app1_fig2

  • Relative Phosphorylation Efficiency:  fp% = (IY-IA) / (IpY-IA)*100
  • IY – signal intensity of phosphorylated Y-peptide
  • IpY – intensity of synthetic phosphopeptide
  • IA – intensity of Y>A substitution peptide as negative control