ac401415z_si_004.xls (46 kB)
Isotope-Coded ATP Probe for Quantitative Affinity Profiling of ATP-Binding Proteins
dataset
posted on 2013-08-06, 00:00 authored by Yongsheng Xiao, Lei Guo, Yinsheng WangATP-binding proteins play significant
roles in numerous cellular
processes. Here, we introduced a novel isotope-coded ATP-affinity
probe (ICAP) as an acylating agent to simultaneously enrich and incorporate
isotope label to ATP-binding proteins. By taking advantage of the
quantitative capability of this isotope-coded probe, we devised an
affinity profiling strategy to comprehensively characterize ATP–protein
interactions at the entire proteome scale. False-positive identification
of ATP-binding sites derived from nonspecific labeling was effectively
minimized through the comparison of the labeling behaviors of lysine
residues with the use of low and high concentrations of the ICAP reagents.
A total of 258 previously known ATP-binding proteins from lysates
of HeLa-S3 and Jurkat-T cells were validated with this affinity profiling
assay. Additionally, we demonstrated that this novel quantitative
ATP-affinity profiling strategy is particularly useful for unveiling
previously unrecognized nucleotide-binding sites in ATP-binding proteins.
For example, our profiling results revealed K356 as a new ATP-binding
site in HSP90. Furthermore, 293 proteins without documented ATP-binding
GO were predicted to be ATP-binding proteins on the basis of our quantitative
affinity profiling results. We also uncovered, for the first time,
the ATP-binding capability of human proliferating cell nuclear antigen
(PCNA), identified the lysine residue involved in ATP binding, and
validated the protein’s capacity in ATP binding with an independent
assay. The ICAP approach described in the present paper should be
generally applicable for the quantitative assessment of ATP-binding
proteins in proteomic samples from cells and tissues.