title: Isotope-coded ATP probe for quantitative affinity profiling of ATP-binding proteins.
abstract: ATP-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.
authors.affiliation: Department of Chemistry, University of California, Riverside, California 92521-0403, United States.
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Xiao
authors.forename: Lei
authors.initials: L
authors.lastname: Guo
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D006367:HeLa Cells; D006801:Humans; D007554:Isotopes; D008958:Models, Molecular; D015335:Molecular Probes / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D007554:Isotopes; D015335:Molecular Probes; D020543:Proteome; D000255:Adenosine Triphosphate