Proteome-Wide Discovery of Unknown ATP-Binding Proteins and Kinase Inhibitor Target Proteins Using an ATP Probe

ATP-binding proteins, including protein kinases, play essential roles in many biological and pathological processes and thus these proteins are attractive as drug targets. Acyl-ATP probes have been developed as efficient probes for kinase enrichment, and these probes have also been used to enrich other ATP-binding proteins. However, a robust method to identify ATP-binding proteins with systematic elimination of nonspecific binding proteins has yet to be established. Here, we describe an ATP competition assay that permitted establishment of a rigorous ATP-binding protein list with virtual elimination of nonspecific proteins. A total of 539 ATP-binding protein candidates were identified, including 178 novel candidates. In informatics analysis, ribosomal proteins were overrepresented in the list of novel candidates. We also found multiple ATP-competitive sites for several kinases, including epidermal growth factor receptor, serine/threonine-protein kinase PRP4 homologue, cyclin-dependent kinase 12, eukaryotic elongation factor 2 kinase, ribosomal protein S6 kinase alpha-1, and SRSF protein kinase 1. Using our cataloged ATP-binding protein list, a selectivity profiling method that covers the kinome and ATPome was established to identify off-target binding sites of ATP-competitive kinase inhibitors, staurosporine and crizotinib

Improved Proteome and Phosphoproteome Analysis on a Cation Exchanger by a Combined Acid and Salt Gradient

Currently used elution methods for strong cation exchange (SCX) chromatography are based on two principles: salt and pH gradient. In this paper, we report the first observation of peptide elution by acid gradient. The degree of peptide separation using C18-SCX StageTip was greatly improved by our acid and salt-based elution method compared with a salt-based elution method. This development enabled us to identify over 22 000 phosphopeptides from 2 mg of protein without labor-intensive sample preparation. Our method is simple, robust, scalable, and low-cost and can be easily implemented without any special equipment or techniques

Protein profiling between metastasized tumors of control and SB-431542-treated groups.

<p>(A) A PCA plot based on the peptide profile in control and SB-431542-treated tumors using SMCA13 for the multivariate analysis and (B) corresponding loading plot. (C) OPLS-DA plot discriminates proteins from those two groups. Black dots represent the control group (n = 4) and white dots represent the SB-431542-treated group (n = 4). (D) S-plot shows the significance of protein variations between control and SB-431542-treated groups. Each black dot represents individual proteins. Black dots in the positive directions indicate the decreased proteins in SB-431542-treated tumors. The increased proteins in SB-431542-treated tumors are presented as dots in the negative direction. Vimentin (Vim), Hsp90ab1 and Eno1 were double-line-circled as the top proteins of the highest confidence and greatest contribution separation between the untreated and SB-431542-treated mice. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126483#pone.0126483.s005" target="_blank">S1 Table</a>). A magnified picture of the significantly reduced proteins in SB-431542-treated tumors is shown (p(corr) > 0.80).</p

Orthotopic metastasis model demonstrates that a specific inhibitor of TGF-β receptor kinase, SB-431542, decreases lung metastasis but does not significantly alter growth of the primary tumor.

<p>(A) Scheme for the experimental approach using 4T1 metastasis model. 4T1 tumor cells (1×10⁴ cells) were transplanted into the mammary fat pad of Balb/c mice. Mice bearing 4T1 mammary tumors were treated three times weekly with SB-431542 (10 mg/kg body weight) or vehicle (20% DMSO/80% corn oil). At day 10 post-injection of 4T1 cells, primary tumors were surgically excised, and the mice were kept alive to allow the tumor to metastasize to the lung. (B) Graph showing relative primary tumor growth of 4T1 cells over time. Data are presented as mean ± SEM. (ns = not significant, control, n = 14; SB-431542, n = 15, unpaired <i>t</i>-test). (C) The number of gross metastasis (control, n = 14; SB-431542-treated, n = 15) was counted. The administration of SB-431542 markedly reduced both the number and the size of the metastasized tumor compared to the control group. Lungs were collected after 40 days and the lung surface was examined for the metastasis. The number of visible lung metastasis was counted. ** p<0.001.; Mann Whitney <i>U</i>-test. Data are presented as the median ± SEM. (D) Representative gross lung images from control and SB-431542-treated groups are shown from control (top) and SB-431542-treated (bottom) animals, with metastases visible at the lung surface marked by bold black arrows. (E) Representative histological view of lung metastases treated with vehicle, DMSO (left) and SB-431542 (right). H&E staining; black dotted lines demarcate tumor parenchyma (*) from the normal lung tissue.</p

Validation of protein expression using western blot and immunohistochemistry.

<p>(A) Metastases lysates were prepared from three mice per group and evaluated by western blot with anti-vimentin, anti-Hsp90a/b, and anti-Eno1 antibodies. β-actin was used as internal normalization. (B) Representative image of positive vimentin staining is shown exclusively in the metastases lesion and not in the surrounding lung tissue. White dotted lines represent a boundary of tumor and surrounding normal lung tissue. M, metastases; NT, normal lung tissue; PV, pulmonary vein. (C) Randomly selected high power fields were immunostained with vimentin and were quantitated using Zeiss software with the % area occupied by metastasized nodules in 4T1 metastasized tumors. A significant reduction in the number of vimentin positive cells was seen in the SB-431542-treated tumors. Scale bars represent 100 μm; hpf, high power fields.</p

Protein expression profiling of 4T1 metastases tumor using dimethyl labeling with triplex stable isotopes.

<p>(A) Schematic representation of the proteomics approach. Lung-metastasized 4T1 tumor samples were isolated from each group (control and SB-431542-treated tumors). Subsequently, the tumor protein was lysed, and differential protein expression was detected by relative quantification using dimethyl-labeling (light, medium and high) followed by an SCX-LC-MS/MS (LTQ Orbitrap Velos) analysis for each set of sample quadruplicates. Isotope-light was used as the reference sample and contained mixed aliquots of all control and SB-431542-treated tumor samples. Four sets of dimethyl-labeled experiments were performed to compare the protein profiles of 8 metastasized tumor samples. (B) Representative dimethyl labeled-based LC-MS/MS spectrum for one of the peptides from vimentin (P20152) showing RQVQSLTCEVDALK, a double charged peptide.</p

EIF signaling was the most enriched canonical IPA pathway of the down-regulated proteins in SB-431542-treated groups.

<p>(A) Pathway analysis conducted using IPA (<a href="http://www.ingenuitypathway.com" target="_blank">www.ingenuitypathway.com</a>) showed a ranking of the most enriched pathways from the down-regulated proteins in SB-431542-treated groups. (B) Whole cell lysates from each group were evaluated by western blot with the EIF family of proteins: anti-eIF4G1, anti-eIF4E, and anti-Eef2 antibodies. (n = 3/group).β -actin was used as a loading control. (C) Sections were assessed by H&E staining (top panel), and immunohistochemistry was performed using anti-eIF4G1 (middle panel) and anti-eIF4E (bottom panel) antibodies. Both eIF4G1 and eIF4E immunostaining in 4T1 lung metastasis showed the heterogeneity of staining patterns seen among individual metastases. 100× magnification. Tumor sections are boxed with a black dotted line, M, metastases; NT, normal lung tissue. (D) eIF4G1- and eIF4E-positive areas were semiquantified using Image Pro Premier Software in three randomly selected high power fields from each samples. Data are represented as box plots (n = 3/group), ** p<0.001 using an unpaired <i>t</i>-test.</p