Site-specific, covalent attachment of poly(dT)-modified peptides to solid surfaces for microarrays.
The present study reported proof-of-principle for a kinase assay approach that can detect specific peptide phosphorylation events. The method involves attachment of peptides onto commercial aminosilane and polycarbodiimide-coated glass slides, using a newly developed DNattach linker system that consists of a poly(dT) tail (Nisshinbo Industries Inc.), followed by a detection step using fluorescently labeled antiphosphoamino acid antibodies. The linker-modified peptides are efficiently synthesized by Michael addition between maleimido-modified peptides and thiol-containing DNattach. Specific covalent immobilization of the modified peptides onto aminosilane and poly carbodiimide-coated slides is then achieved by short exposure to UV-light. Highly selective and quantitative recognition by standard antiphosphoamino acid antibodies (antiphosphotyrosine and anti-phosphoGFAP) and kinases (c-Src and PKA) to the corresponding modified peptides on the microarray spots is demonstrated. Furthermore, we found that this immobilization method provides greater signal-to-noise ratio and better discrimination ability of phosphorylated amino acids than does the conventional immobilization technique. The phosphorylation pattern of target sequences, detected using fluorescently labeled antiphosphoamino acid antibodies, revealed that the linker system preference of the kinase is determined by its activity profile.
Kimura
Naoki
N
Research and Development Center, Nisshinbo Industries Inc, Chiba, Japan. n-kimu@nisshinbo.co.jp
Okegawa
Takashi
T
Yamazaki
Kiyokazu
K
Matsuoka
Koji
K
D000586:Amination; D000081247:CSK Tyrosine-Protein Kinase; D002234:Carbodiimides / Q000737:chemistry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005898:Glass / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011071:Poly T / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012680:Sensitivity and Specificity; D012821:Silanes / Q000737:chemistry; D013499:Surface Properties; D020032:Tyrphostins / Q000494:pharmacology; D014466:Ultraviolet Rays; D019061:src-Family Kinases
D002234:Carbodiimides; D010455:Peptides; D047428:Protein Kinase Inhibitors; D012821:Silanes; D020032:Tyrphostins; C060152:tyrphostin 47; D011071:Poly T; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D017868:Cyclic AMP-Dependent Protein Kinases