Proteome-Wide Discovery and Characterizations of Nucleotide-Binding Proteins with Affinity-Labeled Chemical Probes

Abstract

Nucleotide-binding proteins play pivotal roles in many cellular processes including cell signaling. However, targeted study of sub-proteome of nucleotide-binding proteins, especially protein kinases and GTP-binding proteins, remained challenging. Here, we reported a general strategy in using affinity-labeled chemical probes to enrich, identify, and quantify ATP- and GTP-binding proteins in the entire human proteome. Our results revealed that the ATP/GTP affinity probes facilitated the identification of 100 GTP-binding proteins and 206 kinases with the use of low mg quantities of lysate of HL-60 cells. In combination with the use of SILAC-based quantitative proteomics method, we assessed the ATP/GTP binding selectivities of nucleotide-binding proteins at the global proteome scale. Our results confirmed known and, more importantly, unveiled new ATP/GTP-binding preferences of hundreds of nucleotide-binding proteins. Additionally, our strategy led to the identification of three and one unique nucleotide-binding motifs for kinases and GTP-binding proteins, respectively, and the characterizations of the nucleotide binding selectivities of individual motifs. Our strategy for capturing and characterizing ATP/GTP-binding proteins should be generally applicable for those proteins that can interact with other nucleotides.

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

“…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).…”

“…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.…”

“…To improve the specificity of our MRM-based kinome assay, these targeted peptides in our final kinome library were further divided into two categories depending on the local amino acid sequences surrounding the desthiobiotin-modified lysine. In the category with the highest confidence (class I), target peptides represent the confirmed ATP-binding sites on kinases and must satisfy one of the following two criteria: (A) Peptides cover at least one of the three ATP-binding motifs; discovered in our previous studies, i.e., HRDxKxxN, VAxK or GxxxxGK (15,21); (B) Peptides contain the ATP-binding sites discovered in our previous ATP-affinity profiling assay even though they do not reside in any of the conserved motif sequences (21). For instance, although FLSGLELVK#QGAEAR (K# represents the probe-labeled lysine), a peptide from TP53-regulating kinase, does not belong to any of the aforementioned conserved motifs, it displays strong ATP binding affinity in our previous report (21).…”

“…Recently, we and others reported the application of biotinconjugated acyl nucleotide probes for the enrichment and identification of kinases from complex protein mixtures (13)(14)(15)(16)(17). This enrichment technique, in combination with multidimensional LC-MS platform, facilitates the identification of ϳ200 protein kinases (15).…”

“…Recently, we and others reported the application of biotinconjugated acyl nucleotide probes for the enrichment and identification of kinases from complex protein mixtures (13)(14)(15)(16)(17). This enrichment technique, in combination with multidimensional LC-MS platform, facilitates the identification of ϳ200 protein kinases (15). Despite these advances, such large-scale kinome studies are often performed in the datadependent acquisition (DDA) mode, where typically 10 -20 most abundant ions found in MS are subsequently selected for fragmentation in MS/MS to enable peptide identification (18).…”