Yongsheng Xiao

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

Kinases are among the most intensively pursued enzyme superfamilies as targets for anti-cancer drugs. Large data sets on inhibitor potency and selectivity for more than 400 human kinases became available recently, offering the opportunity to design rationally novel kinase-based anti-cancer therapies. However, the expression levels and activities of kinases are highly heterogeneous among different types of cancer and even among different stages of the same cancer. The lack of effective strategy for profiling the global kinome hampers the development of kinase-targeted cancer chemotherapy. Here, we introduced a novel global kinome profiling method, based on our recently developed isotope-coded ATP-affinity probe and a targeted proteomic method using multiple-reaction monitoring (MRM), for assessing simultaneously the expression of more than 300 kinases in human cells and tissues. This MRM-based assay displayed much better sensitivity, reproducibility, and accuracy than the discovery-based shotgun proteomic method. Approximately 250 kinases could be routinely detected in the lysate of a single cell line. Additionally, the incorporation of iRT into MRM kinome library rendered our MRM kinome assay easily transferrable across different instrument platforms and laboratories. We further employed this approach for profiling kinase expression in two melanoma cell lines, which revealed substantial kinome reprogramming during cancer progression and demonstrated an excellent correlation between the anti-proliferative effects of kinase inhibitors and the expression levels of their target kinases. Therefore, this facile and accurate kinome profiling assay, together with the kinome-inhibitor interaction map, could provide invaluable knowledge to predict the effectiveness of kinase inhibitor drugs and offer the opportunity for individualized cancer chemotherapy.

Proteome-wide Discovery and Characterizations of Nucleotide-binding Proteins with Affinity-labeled Chemical Probes

Nucleotide-binding proteins play pivotal roles in many cellular processes including cell signaling. However, targeted studies of the subproteome of nucleotide-binding proteins, especially protein kinases and GTP-binding proteins, remain challenging. Here, we report 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 milligram quantities of lysate of HL-60 cells. In combination with the use of the stable isotope labeling by amino acids in cell culture-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.

Isotope-coded ATP Probe for Quantitative Affinity Profiling of ATP-binding Proteins

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 proteins 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.

Profiling global kinome signatures of the radioresistant MCF-7/C6 breast cancer cells using MRM-based targeted proteomics.

Ionizing radiation is widely used in cancer therapy; however, cancer cells often develop radioresistance, which compromises the efficacy of cancer radiation therapy. Quantitative assessment of the alteration of the entire kinome in radioresistant cancer cells relative to their radiosensitive counterparts may provide important knowledge to define the mechanism(s) underlying tumor adaptive radioresistance and uncover novel target(s) for effective prevention and treatment of tumor radioresistance. By employing a scheduled multiple-reaction monitoring analysis in conjunction with isotope-coded ATP affinity probes, we assessed the global kinome of radioresistant MCF-7/C6 cells and their parental MCF-7 human breast cancer cells. We rigorously quantified 120 kinases, of which 1/3 exhibited significant differences in expression levels or ATP binding affinities. Several kinases involved in cell cycle progression and DNA damage response were found to be overexpressed or hyperactivated, including checkpoint kinase 1 (CHK1), cyclin-dependent kinases 1 and 2 (CDK1 and CDK2), and the catalytic subunit of DNA-dependent protein kinase. The elevated expression of CHK1, CDK1, and CDK2 in MCF-7/C6 cells was further validated by Western blot analysis. Thus, the altered kinome profile of radioresistant MCF-7/C6 cells suggests the involvement of kinases on cell cycle progression and DNA repair in tumor adaptive radioresistance. The unique kinome profiling results also afforded potential effective targets for resensitizing radioresistant cancer cells and counteracting deleterious effects of ionizing radiation exposure.

Quantitative Proteomic Analysis Revealed Lovastatin-induced Perturbation of Cellular Pathways in HL-60 Cells

Lovastatin, a member of the statin family of drugs, is widely prescribed for treating hypercholesterolemia. The statin family of drugs, however, also shows promise for cancer treatment and prevention. Although lovastatin is known to be an inhibitor for HMG-CoA reductase, the precise mechanisms underlying the drugs antiproliferative activity remain unclearly defined. Here we utilized mass spectrometry, in conjunction with stable isotope labeling by amino acids in cell culture (SILAC), to analyze the perturbation of protein expression in HL-60 cells treated with lovastatin. We were able to quantify ∼3200 proteins with both forward and reverse SILAC labeling experiments, among which ∼120 exhibited significant alterations in expression levels upon lovastatin treatment. Apart from confirming the expected inhibition of the cholesterol biosynthesis pathway, our quantitative proteomic results revealed that lovastatin perturbed the estrogen receptor signaling pathway, which was manifested by the diminished expression of estrogen receptor α, steroid receptor RNA activator 1, and other related proteins. Lovastatin also altered glutamate metabolism through down-regulation of glutamine synthetase and γ-glutamylcysteine synthetase. Moreover, lovastatin treatment led to a marked down-regulation of carbonate dehydratase II (a.k.a. carbonic anhydrase II) and perturbed the protein ubiquitination pathway. Together, the results from the present study underscored several new cellular pathways perturbed by lovastatin.

Global discovery of protein kinases and other nucleotide‐binding proteins by mass spectrometry

Nucleotide-binding proteins, such as protein kinases, ATPases and GTP-binding proteins, are among the most important families of proteins that are involved in a number of pivotal cellular processes. However, global study of the structure, function, and expression level of nucleotide-binding proteins as well as protein-nucleotide interactions can hardly be achieved with the use of conventional approaches owing to enormous diversity of the nucleotide-binding protein family. Recent advances in mass spectrometry (MS) instrumentation, coupled with a variety of nucleotide-binding protein enrichment methods, rendered MS-based proteomics a powerful tool for the comprehensive characterizations of the nucleotide-binding proteome, especially the kinome. Here, we review the recent developments in the use of mass spectrometry, together with general and widely used affinity enrichment approaches, for the proteome-wide capture, identification and quantification of nucleotide-binding proteins, including protein kinases, ATPases, GTPases, and other nucleotide-binding proteins. The working principles, advantages, and limitations of each enrichment platform in identifying nucleotide-binding proteins as well as profiling protein-nucleotide interactions are summarized. The perspectives in developing novel MS-based nucleotide-binding protein detection platform are also discussed.

Hexavalent chromium-induced alteration of proteomic landscape in human skin fibroblast cells.

Hexavalent chromium [Cr(VI)] generated during industrial processes is carcinogenic. Although much is known about the deleterious effects caused by reactive oxygen species generated during the reduction of Cr(VI) after its absorption by biological systems, the precise mechanisms underlying Cr(VI) cytotoxicity remain poorly defined. Here, we analyzed, at the global proteome scale, the perturbation of protein expression in GM00637 human skin fibroblast cells upon exposure to potassium dichromate (K₂Cr₂O₇). We were able to quantify ∼4600 unique proteins, among which ∼400 exhibited significant alterations in expression levels upon a 24-h treatment with 0.5 μM K₂Cr₂O₇. Pathway analysis revealed the Cr(VI)-induced perturbation of cholesterol biosynthesis, G-protein signaling, inflammatory response, and selenoprotein pathways. In particular, we discovered that the K₂Cr₂O₇ treatment led to pronouncedly elevated expression of a large number of enzymes involved in de novo cholesterol biosynthesis. Real-time PCR analysis revealed the increased mRNA expression of selected genes involved in cholesterol biosynthesis. Consistently, K₂Cr₂O₇ treatment resulted in marked increases in cellular cholesterol level in multiple cell lines. Moreover, the Cr(VI)-induced growth inhibition of cultured human cells could be rescued by a cholesterol-lowering drug, lovastatin. Together, we demonstrated, for the first time, that Cr(VI) may exert its cytotoxic effect, at least partly, through the up-regulation of enzymes involved in de novo cholesterol biosynthesis and the resultant increase of cholesterol level in cells.

Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ~6500 unique proteins quantified, ~300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content.

Application of Adenosine Triphosphate Affinity Probe and Scheduled Multiple-Reaction Monitoring Analysis for Profiling Global Kinome in Human Cells in Response to Arsenite Treatment

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

Comprehensive Characterization of SGTP-Binding Proteins by Orthogonal Quantitative SGTP-Affinity Profiling and SGTP/GTP Competition Assays

Thiopurine drugs are widely used as antileukemic drugs and immunosuppressive agents, and 6-thioguanosine triphosphate (SGTP) is a major metabolite for these drugs. Recent studies have suggested that thiopurine drugs may exert their cytotoxic effects partly through binding of SGTP to a GTP-binding protein, Rac1. However, it remains unclear whether SGTP can also bind to other cellular proteins. Here, we introduced an orthogonal approach, encompassing nucleotide-affinity profiling and nucleotide-binding competition assays, to characterize comprehensively SGTP-binding proteins along with the specific binding sites from the entire human proteome. With the simultaneous use of SGTP and GTP affinity probes, we identified 165 SGTP-binding proteins that are involved in several different biological processes. We also examined the binding selectivities of these proteins toward SGTP and GTP, which allowed for the revelation of the relative binding affinities of the two nucleotides toward the nucleotide-binding motif sequence of proteins. Our results suggest that SGTP mainly targets GTPases, with strong binding affinities observed for multiple heterotrimeric G proteins. We also demonstrated that SGTP binds to several cyclin-dependent kinases (CDKs), which may perturb the CDK-mediated phosphorylation and cell cycle progression. Together, this represents the first comprehensive characterization of SGTP-binding property for the entire human proteome. We reason that a similar strategy can be generally employed for the future characterization of the interaction of other modified nucleotides with the global proteome.