Design Example

  • First we compiled a list of known substrate peptides. (Shown in Table 1)
Table 1
PK Names PK Types Number of Substrate Peptides
EFGR Tyrosine Protein Kinase 56
SRC Tyrosine Protein Kinase 99
SRC (Mass Analysis) Tyrosine Protein Kinase 157
PKA Ser/Thr Protein Kinase 249
CDK/CDK1 Ser/Thr Protein Kinase 153
ATM, MAPK, Casein II, Cam II Ser/Thr Protein Kinase 298
  • Next we used WebLogo to create sequences logos for each of these protein kinases.
  • The height of the letter is proportional to the probability of a specific amino acid at that particular position.
  • Important – Note that there is no information on sequence preference in the WebLogo display since the probability of an amino acid at a position is independent of the adjacent positions.
  • Figure 1 is the sequence logo for the protein kinase EGFR./li>
Figure 1

  • Next we listed the six most probable amino acids on each side of the phosphorylation site (Table 2).
  • Peptide sequence is numbered from N- (minus number) to C- (positive #) terminal and the phosphorylation site is 0.
Table 2
Amino Acid Position EGFR SRC SRC(3T3) PKA CDK ATM
-7 TDEIAS RLPVES KAEPRL SKGEPT
-6 GESADT DESLKP KRAGSP SGPELA ESDKLP
-5 ADLSNP APESIT XPLSET RLKSAE PSGNEA SDEGLK
-4 EDGPAF ESGPTN SDPESG SKRLEL SPDLGE SEDGTL
-3 EDGNPK EDSPGN EDSAXG RKTP TPLGSA RESLGD
-2 DPSAEV PDGENA DEGASN RKSTQ PSALTF SEPDLQ
-1 DEILNR VITAEQ LIPTAD LSRAPG SGPLTK SLDEAV
0 Y Y Y ST ST S
+1 VIELQN EDSAGV SDEGAT LSVRFG P DQEPSG
+2 EQNDSI AESVDK LAGDEA SAPTDL SPRVAG EDSGPL
+3 PVAFQL PVLAIM LAGDES SEAGLP KRPSGL EDSAGL
+4 PSTFEN EAVGKD XPGSDQ LSEPRQ SKPARL EDSGPI
+5 PEKALD GAKSVF SPGSKE SALRET SPRGTA EDSLAK
+6 SGNDLQ GQSPEY XRGSPK SALRET ASRTGL EDSGLA
+7 PKSRMF KEGLSD ASKRDE PRLKTA
  • Next we put together some additional design considerations.
  • R to represent R and K and S to represent S and T.
  • Additional AA (not listed in Table 2) are incorporated for exploratory purposes.
  • The synthesis of the pS-containing peptides (at position 0) provides reference in quantitative phosphorylation measurements.
  • The concentration = variable design generates a peptide pico-titer plate in a microarray format and a large number of enzymatic reaction curves can be measured simultaneously.
  • We can use these considerations along with the information in Table 2 to generate up to thousands of heptamer peptides per chip.
  • Table 3 and 4 represent 2 different possible chip designs.
Table 3
PKS – Peptide Microarray – Chip 1 (Peptide Concentration = Constant)
Amino Acid Position in Peptide Amino Acid to be Synthesized No. of Amino Acids at this Position
-3 R 1
-2 RSQPALF 7
-1 LSRAPK 6
0 pSSTAY 5
+1 LSVRFG 6
+2 A 1
+3 E 1
No. of Seq/Set 1,260
No. Conc. Sites 1
Total No. of Sequences 1,260
No. of Replicates 3
Total No. of Sequences with Replicates 3,780
Table 4
PKS – Pico-Titer Plate Peptide Microarray – Chip 2 (Peptide Concentration = Variable)
Amino Acid Position in Peptide Amino Acid to be Synthesized No. of Amino Acids at this Position
-3 R 1
-2 RSQPLF 6
-1 LSRAP 5
0 pSS 2
+1 LF 2
+2 A 1
+3 E 1
No. of Seq/Set 120
No. Conc. Sites 10
Total No. of Sequences 1,200
No. of Replicates 3
Total No. of Sequences with Replicates 3,600
  • The design of the peptide sequences is flexible to different peptide sequence lengths and residue choices.
  • For exploratory or preliminary experiments, the number of replicates may be reduced to allow a larger number of peptides examined.