Development and validation of a method for profiling post-translational modification activities using protein microarrays.
BACKGROUND
Post-translational modifications (PTMs) impact on the stability, cellular location, and function of a protein thereby achieving a greater functional diversity of the proteome. To fully appreciate how PTMs modulate signaling networks, proteome-wide studies are necessary. However, the evaluation of PTMs on a proteome-wide scale has proven to be technically difficult. To facilitate these analyses we have developed a protein microarray-based assay that is capable of profiling PTM activities in complex biological mixtures such as whole-cell extracts and pathological specimens.


RESULTS
In our assay, protein microarrays serve as a substrate platform for in vitro enzymatic reactions in which a recombinant ligase, or extracts prepared from whole cells or a pathological specimen is overlaid. The reactions include labeled modifiers (e.g., ubiquitin, SUMO1, or NEDD8), ATP regenerating system, and other required components (depending on the assay) that support the conjugation of the modifier. In this report, we apply this methodology to profile three molecularly complex PTMs (ubiquitylation, SUMOylation, and NEDDylation) using purified ligase enzymes and extracts prepared from cultured cell lines and pathological specimens. We further validate this approach by confirming the in vivo modification of several novel PTM substrates identified by our assay.


CONCLUSIONS
This methodology offers several advantages over currently used PTM detection methods including ease of use, rapidity, scale, and sample source diversity. Furthermore, by allowing for the intrinsic enzymatic activities of cell populations or pathological states to be directly compared, this methodology could have widespread applications for the study of PTMs in human diseases and has the potential to be directly applied to most, if not all, basic PTM research.
Del Rincón
Sonia V
SV
Signal Transduction Program, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
Rogers
Jeff
J
Widschwendter
Martin
M
Sun
Dahui
D
Sieburg
Hans B
HB
Spruck
Charles
C
D006367:HeLa Cells; D006801:Humans; D000075687:NEDD8 Protein; D040081:Protein Array Analysis; D011499:Protein Processing, Post-Translational; D020543:Proteome; D025842:SUMO-1 Protein / Q000378:metabolism; D054875:Ubiquitination; D014452:Ubiquitins / Q000378:metabolism
D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D020543:Proteome; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D014452:Ubiquitins