array consisting of 1176 substrates  (Pepscan Inc)

Journal: PLoS ONE

Article Title: Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

doi: 10.1371/journal.pone.0006440

Figure Lengend Snippet: A. Plot showing the phosphorylation of various substrates at 1, 5, 10, 20, 30, 60 & 120 min. time points. B. Time coefficient plotted against intensity. C. Images of trial arrays at 1 minute, 30 minute and 120 minute time-points showing the steady increase in phosphorylation level. An overlay for array orientation is also shown.

Article Snippet: The array consisting of 1176 substrates was purchased from Pepscan systems (Lelystad, The Netherlands) and the design is described in detail on their website: http://www.pepscanpresto.com/index.php?id=27 .

Techniques: Phospho-proteomics

Journal: PLoS ONE

Article Title: Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

doi: 10.1371/journal.pone.0006440

Figure Lengend Snippet: A. In vitro phosphorylation of MEK by c-Raf. The capacity of purified c-Raf for in vitro phosphorylation studies was examined by incubating purified c-Raf with MEK and detected using MEK Ser218/222 /MEK2 Ser222/226 antibodies. B. c-Raf phosphorylation of 1176 peptide array. Phosphorylation of the 1176 peptide array, spotted in duplicate, on incubation with c-Raf and 33 P-γ-ATP for one hour shows differential phosphorylation of the various substrate peptides demonstrating that peptide sequences confer specificity to c-Raf-dependent phosphorylation. Further analysis was carried out to determine whether the primary sequence of the peptides employed revealed information as to the amino acid preferences of this enzyme for substrate phosphorylation. C. Consensus sequence of c-Raf substrates using 1176 array design. Consensus sequence determined for c-Raf substrates on analysis of peptide array data shows a strong selection for both hydrophobic and basic residues at the −3 position. A strong preference for leucine is seen at the −2 position. Proline and arginine are strongly preferred at the −1 position. An examination of the amino acid preferences C-terminal to the fixed phosphorylated residue reveals a bias towards aspargine compared to other residues at the +1 position. Also, acyclic and hydrophobic amino acids are seen at the +1 position with no preference for any particular group of amino acid at the +2 position. The +3 position shows a strong preference for basic residues.

Article Snippet: The array consisting of 1176 substrates was purchased from Pepscan systems (Lelystad, The Netherlands) and the design is described in detail on their website: http://www.pepscanpresto.com/index.php?id=27 .

Techniques: In Vitro, Phospho-proteomics, Purification, Peptide Microarray, Incubation, Sequencing, Selection, Residue

Journal: PLoS ONE

Article Title: Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

doi: 10.1371/journal.pone.0006440

Figure Lengend Snippet: A. c-Raf phosphorylation of 1024 peptide array. Phosphorylation of the custom made 1024 peptide array, spotted in triplicate, on incubation with c-Raf and 33 P-γ-ATP for one hour shows differential phosphorylation of the various substrate peptides. B. Consensus sequence of c-Raf substrates using 1024 array design. Consensus sequence obtained from the 1024 peptide array shows a strong preference for arginine at −1,−4 and the −5 position while the −2 position shows a strong preference for a hydrophobic residue and no distinctive preference is seen at the −3 position. Analysis of the C-terminal position relative to the centrally fixed phosphorylated residue shows a very high preference for methionine besides an equal preference for other basic and hydrophobic amino acids at the +1 position. Arginine is preferred at the +2 and +4 positions while methionine and proline along with arginine are preferred at +3 positions. Hydrophobic residues are preferred over basic residues at +5.

Article Snippet: The array consisting of 1176 substrates was purchased from Pepscan systems (Lelystad, The Netherlands) and the design is described in detail on their website: http://www.pepscanpresto.com/index.php?id=27 .

Techniques: Phospho-proteomics, Peptide Microarray, Incubation, Sequencing, Residue

Journal: PLoS ONE

Article Title: Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

doi: 10.1371/journal.pone.0006440

Figure Lengend Snippet: A. Alignment of c-raf against MAP3K8. Alignment of c-Raf with MAP3K8 using BLAST shows considerable homology between their kinase domains. B. Phosphorylation by MAP3K8 of 1176 peptide array. Specific incorporation of radioactivity into different peptides was observed after incubating the 1176 peptide array with MAP3K8 and 33 P-γ-ATP. C. Comparison of c-Raf and MAP3K8 peptide phosphorylation. A correlation plot between substrate phosphorylation of c-Raf and MAP3K8 (using the 1176 peptide array design), which indicates that despite the highly similar primary sequences both enzymes have different substrate preferences. D. Consensus sequence of MAP3K8 substrates using 1176 array design. Consensus sequence for MAP3K8 shows preferences for phenylalanine at −3, −2, −1 and +1 positions and lysine at all six positions, which is similar to c-raf that has a preference for arginine instead of lysine at all positions as shown in . E. Flow chart depicting the MAPK cascade and the roles of various kinases including c-Raf and MAP3K8 within these cascades.

Article Snippet: The array consisting of 1176 substrates was purchased from Pepscan systems (Lelystad, The Netherlands) and the design is described in detail on their website: http://www.pepscanpresto.com/index.php?id=27 .

Techniques: Phospho-proteomics, Peptide Microarray, Radioactivity, Comparison, Sequencing