Nano-flow liquid chromatography tandem mass spectrometry (nano-flow LC-MS/MS) is the mainstay in proteome research because of its excellent sensitivity but often comes at the expense of robustness. Here we show that micro-flow LC-MS/MS using a 1 × 150 mm column shows excellent reproducibility of chromatographic retention time (<0.3% coefficient of variation, CV) and protein quantification (<7.5% CV) using data from >2000 samples of human cell lines, tissues and body fluids. Deep proteome analysis identifies >9000 proteins and >120,000 peptides in 16 h and sample multiplexing using tandem mass tags increases throughput to 11 proteomes in 16 h. The system identifies >30,000 phosphopeptides in 12 h and protein-protein or protein-drug interaction experiments can be analyzed in 20 min per sample. We show that the same column can be used to analyze >7500 samples without apparent loss of performance. This study demonstrates that micro-flow LC-MS/MS is suitable for a broad range of proteomic applications.

Integrated analysis of genomes, transcriptomes, proteomes and drug responses of cancer cell lines (CCLs) is an emerging approach to uncover molecular mechanisms of drug action. We extend this paradigm to measuring proteome activity landscapes by acquiring and integrating quantitative data for 10,000 proteins and 55,000 phosphorylation sites (p-sites) from 125 CCLs. These data are used to contextualize proteins and p-sites and predict drug sensitivity. For example, we find that Progesterone Receptor (PGR) phosphorylation is associated with sensitivity to drugs modulating estrogen signaling such as Raloxifene. We also demonstrate that Adenylate kinase isoenzyme 1 (AK1) inactivates antimetabolites like Cytarabine. Consequently, high AK1 levels correlate with poor survival of Cytarabine-treated acute myeloid leukemia patients, qualifying AK1 as a patient stratification marker and possibly as a drug target. We provide an interactive web application termed ATLANTiC ( http://atlantic.proteomics.wzw.tum.de ), which enables the community to explore the thousands of novel functional associations generated by this work.

Summary The mitotic kinase AURORA-A is essential for cell cycle progression and is considered a priority cancer target. While the catalytic activity of AURORA-A is essential for its mitotic function, recent reports indicate an additional non-catalytic function, which is difficult to target by conventional small molecules. We therefore developed a series of chemical degraders (PROTACs) by connecting a clinical kinase inhibitor of AURORA-A to E3 ligase-binding molecules (e.g. thalidomide). One degrader induced rapid, durable and highly specific degradation of AURORA-A. In addition ,we found that the degrader complex was stabilized by cooperative binding between AURORA-A and CEREBLON. Degrader-mediated AURORA-A depletion caused an S-phase defect, which is not the cell cycle effect observed upon kinase inhibition, supporting an important non-catalytic function of AURORA-A during DNA replication. AURORA-A degradation induced rampant apoptosis in cancer cell lines, and thus represents a versatile starting point for developing new therapeutics to counter AURORA-A function in cancer.

In total, more than 700 proteins regulate chromatin function 18,[22][23] and they are often part of multi-domain protein complexes. Beside the catalytic subunit that controls chromatin accessibility, also subunits that recognize and interact with epigenetic modifications are crucial components of histone modifying complexes. 2 Despite the three classes of epigenetic readers, erasers, and writers, also epigenetic movers, shapers and insulators interact with chromatin structure. [24][25] Proteins that recognize post-translational modifications are classified as epigenetic readers. 26 Well-studied protein families for epigenetic readers are, e.g., bromodomains (BRDs), which recognize acetylated lysine residues. The BRDs have been extensively studied and successfully drugged in cancer treatment. 26 In Table 1, the bromodomain BRD4 of the bromodomain and extraterminal domain (BET) family is listed due to its prominent role in super-enhancers (SEs) organization and regulation of oncogene expression in cancer. 27 Targeting BRD4 by inhibiting the acetyl-lysine binding site with small molecules, e.g., the first BRD targeting inhibitor (JQ1), was shown to be an effective strategy for cancers like the aggressive NUT midline carcinoma (NMC). [28][29] Beside the outstanding role of BRD4, other BRDs are involved as epigenetic readers in various nucleosome remodeling complexes: in the ATP-dependent human complexes BAF (BRG1/BRMassociated factor) and PBAF (polybromo-associated BAF factor), two bromodomains, SMARCA2/ 4 (SWI/SNF-related, matrix-associated actin-dependent regulator of chromatin, subfamily A2/ 4), perturbate with the core subunits BRG1/BRM histone-DNA contacts. [30][31][32] Mutations in BAF components are one of the most frequently observed genetic alteration in cancer. [33][34] demonstrated how mutations and misregulations of histone lysine methyltransferases (KMTs), demethylases and methyl-lysine-binding proteins are connected to various diseases, thus making them effective therapeutic targets for cancer treatments. [46][47] The histone demethylation process is carried out by lysine demethylases like LSD1 48 and the JARID1 familiy 49 epigenetic erasers that are known to be perturbed in cancer, as previously listed in Table 1. Equally involved in cancer formation is the class of histone lysine methyltransferases (KMTs) which are categorized as epigenetic writers. KMTs comprise proteins like MLL1-3 and SET1D which are relevant drug targets, as shown in the non-exhaustive list in Table 1. Within histone lysine methylation, H3K4 methylation is an evolutionary conserved motif that marks active gene transcription 50-51 and is highly enriched at the promotor region and transcription start site. 51 The family of Histone lysine Methyltransferases and its adaptor proteins are described in the following chapter.while the pink colored c-Myc peptide MbIIIb binds to WDR5 on a shallow cleft on the surface, the so called WBM side (pdb entry: 3eg6 and 4y7r).WDR5 has emerged as a promising drug target for anti-cancer therapies as i...

Erythropoietin-producing hepatocellular (EPH) receptors are transmembrane receptor tyrosine kinases. Their extracellular domains bind specifically to ephrin A/B ligands, and this binding modulates intracellular kinase activity. EPHs are key players in bidirectional intercellular signaling, controlling cell morphology, adhesion, and migration. They are increasingly recognized as cancer drug targets. We analyzed the binding of NVP-BHG712 (NVP) to EPHA2 and EPHB4. Unexpectedly, all tested commercially available NVP samples turned out to be a regioisomer (NVPiso) of the inhibitor, initially described in a Novartis patent application. They only differ by the localization of a single methyl group on either one of two adjacent nitrogen atoms. The two compounds of identical mass revealed different binding modes. Furthermore, both in vitro and in vivo experiments showed that the isomers differ in their kinase affinity and selectivity.