High-density peptide microarrays for reliable identification of phosphorylation sites and upstream kinases.
The human genome encodes about 25,000 genes. This number seems to be very small compared to the multitude of different protein functions in highly regulated pathways that are responsible for complex biochemical mechanisms like growth, metabolism, signal transduction and reproduction. Obviously, there are mechanisms creating additional protein diversity. The most important mechanism is post-translational modification (PTM) changing protein surfaces by phosphorylation, sulfation, acetylation, methylation and sumoylation resulting in an about 100-fold higher complexity (1, 2). This chapter presents a very efficient way to detect potential phosphorylation sites in proteins using overlapping peptide scans immobilized on glass slides. Results from 35 different human kinases using peptide microarrays displaying overlapping peptide scans through either all human cyclophilins or all human FK506-binding proteins are shown. Additionally, detection of phosphorylation sites in a proteome-wide manner is demonstrated using peptide microarrays displaying cytomegalovirus proteome in the form of more than 17,000 overlapping peptides.
Thiele
Alexandra
A
Max Planck Research Unit for Enzymology of Protein Folding, Halle, Germany.
Zerweck
Johannes
J
Weiwad
Matthias
M
Fischer
Gunter
G
Schutkowski
Mike
M
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008954:Models, Biological; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011494:Protein Kinases / Q000378:metabolism*; D011868:Radioisotopes / Q000378:metabolism
D010455:Peptides; D011868:Radioisotopes; D011494:Protein Kinases