Sequential multiplex analyte capturing for phosphoprotein profiling.
Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity.
Poetz
Oliver
O
NMI Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstrasse 55, Reutlingen, Germany. poetz@nmi.de
Henzler
Tanja
T
Hartmann
Michael
M
Kazmaier
Cornelia
C
Templin
Markus F
MF
Herget
Thomas
T
Joos
Thomas O
TO
D000818:Animals; D002460:Cell Line; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D018189:Immunomagnetic Separation / Q000295:instrumentation / Q000379:methods; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism
D010750:Phosphoproteins; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases