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 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 non-specific 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.

A Targeted Quantitative Proteomics Strategy for Global Kinome Profiling of Cancer Cells and Tissues

“…Purification-Protein lysates were generated from cultured cells or tissue samples using previously described procedures (see supplementary Materials) (21). HeLa-S3 cells were purchased from the National Cell Culture Center (Minneapolis, MN), and IMR-90, K562, WM-115, and WM-266 -4 cells were obtained from ATCC (Manassas, VA).…”

“…The desthiobiotin-conjugated nucleotide affinity probes were prepared previously (15,21). Approximately 1 mg cell lysate was treated separately with light and heavy labeled desthiobiotin-ATP affinity probes at a final concentration of 100 M. Labeling reactions were carried out with gentle shaking at room temperature for 1.5 h. After the reaction, the remaining probes in the cell lysates were removed by buffer exchange with 25 mM NH 4 HCO 3 (pH 8.5) using Amicon Ultra-4 filters (10,000 NMWL, Millipore).…”

“…The MRM-based kinome assay is built upon our previously described ICAP-based strategy for the simultaneous enrichment and isotopic labeling of ATP-binding proteins in cell lysates (21). The ICAP reagent harbors three components (Fig.…”

“…Peptide Selection-Owing to its relatively high reactivity, the ICAP probe may react with, aside from the lysine residue(s) located at the ATP-binding site, other lysine residues in kinases or other proteins through electrostatic interactions (15,21). The probe labeling emanating from such nonspecific interactions does not reflect the ATP-binding affinities of the kinases.…”

“…Aside from conventional MRM-based assay design, we selectively label and enrich kinases from complex human proteome prior to MRM analysis with the use of desthiobiotin-based isotopecoded ATP-affinity probe (ICAP) (21) to attain high specificity and sensitivity. We demonstrated that this MRM-based kinome detection strategy coupled with ICAP reagent is applicable for clinical samples that are not amenable to metabolic labeling.…”