Development and Discovery of a Selective Degrader of Casein Kinases 1 δ/ε.

Members of the casein kinase 1 (CK1) family have emerged as key regulators of cellular signaling and as potential drug targets. Functional annotation of the 7 human isoforms would benefit from isoform-selective inhibitors, allowing studies on the role of these enzymes in normal physiology and disease pathogenesis. However, due to significant sequence homology within the catalytic domain, isoform selectivity is difficult to achieve with conventional small molecules.

Cell-selective proteomics segregates pancreatic cancer subtypes by extracellular proteins in tumors and circulation.

Cell-selective proteomics is a powerful emerging concept to study heterocellular processes in tissues. However, its high potential to identify non-cell-autonomous disease mechanisms and biomarkers has been hindered by low proteome coverage. Here, we address this limitation and devise a comprehensive azidonorleucine labeling, click chemistry enrichment, and mass spectrometry-based proteomics and secretomics strategy to dissect aberrant signals in pancreatic ductal adenocarcinoma (PDAC).

Time-resolved in vivo ubiquitinome profiling by DIA-MS reveals USP7 targets on a proteome-wide scale.

Mass spectrometry (MS)-based ubiquitinomics provides system-level understanding of ubiquitin signaling. Here we present a scalable workflow for deep and precise in vivo ubiquitinome profiling, coupling an improved sample preparation protocol with data-independent acquisition (DIA)-MS and neural network-based data processing specifically optimized for ubiquitinomics. Compared to data-dependent acquisition (DDA), our method more than triples identification numbers to 70,000 ubiquitinated peptides in single MS runs, while significantly improving robustness and quantification precision.

TOR signaling regulates liquid phase separation of the SMN complex governing snRNP biogenesis.

The activity of the SMN complex in promoting the assembly of pre-mRNA processing UsnRNPs correlates with condensation of the complex in nuclear Cajal bodies. While mechanistic details of its activity have been elucidated, the molecular basis for condensation remains unclear. High SMN complex phosphorylation suggests extensive regulation.

Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation.

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1's error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase.

Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling.

The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling.

Systematic evaluation of label-free and super-SILAC quantification for proteome expression analysis.

Rationale: Advanced implementations of mass spectrometry (MS)-based proteomics allow for comprehensive proteome expression profiling across many biological samples. The outcome of such studies critically depends on accurate and precise quantification, which has to be ensured for high-coverage proteome analysis possible on fast and sensitive mass spectrometers such as quadrupole orbitrap instruments.

Methods: We conducted ultra-high-performance liquid chromatography (UHPLC)/MS experiments on a Q Exactive to systematically compare label-free proteome quantification across six human cancer cell lines with quantification against a shared reference mix generated by stable isotope labeling with amino acids in cell culture (super-SILAC).

Comparative proteome analysis across non-small cell lung cancer cell lines.

Non-small cell lung cancer (NSCLC) cell lines are widely used model systems to study molecular aspects of lung cancer. Comparative and in-depth proteome expression data across many NSCLC cell lines has not been generated yet, but would be of utility for the investigation of candidate targets and markers in oncogenesis. We employed a SILAC reference approach to perform replicate proteome quantifications across 23 distinct NSCLC cell lines.

Quantitative proteomics of kinase inhibitor targets and mechanisms.

Small molecule inhibitors of protein kinases are key tools for signal transduction research and represent a major class of targeted drugs. Recent developments in quantitative proteomics enable an unbiased view on kinase inhibitor selectivity and modes of action in the biological context. While chemical proteomics techniques utilizing quantitative mass spectrometry interrogate both target specificity and affinity in cellular extracts, proteome-wide phosphorylation analyses upon kinase inhibitor treatment identify signal transduction pathway and network regulation in an unbiased manner.

Phosphoregulation of the human SMN complex.

The survival motor neuron (SMN) complex is a macromolecular machine comprising 9 core proteins: SMN, Gemins2-8 and unrip in vertebrates. It performs tasks in RNA metabolism including the cytoplasmic assembly of spliceosomal small nuclear ribonucleoprotein particles (snRNPs). The SMN complex also localizes to the nucleus, where it accumulates in Cajal Bodies (CB) and may function in transcription and/or pre-mRNA splicing.

Comparison of SILAC and mTRAQ quantification for phosphoproteomics on a quadrupole orbitrap mass spectrometer.

Advances in mass spectrometric methodology and instrumentation have promoted a continuous increase in analytical performance in the field of phosphoproteomics. Here, we employed the recently introduced quadrupole Orbitrap (Q Exactive) mass spectrometer for quantitative signaling analysis to a depth of more than 15 000 phosphorylation sites. In parallel to the commonly used SILAC approach, we evaluated the nonisobaric chemical labeling reagent mTRAQ as an alternative quantification technique.

Proteome-wide analysis of temporal phosphorylation dynamics in lysophosphatidic acid-induced signaling.

Most growth factor receptors trigger phosphorylation-based signal transduction to translate environmental stimuli into defined biological responses. In addition to comprehensive and reliable assessment of growth factor-induced phosphoregulation, temporal resolution is needed to gain insights into the organizing principles of the cellular signaling machinery. Here, we introduce a refined experimental design for MS-based phosphoproteomics to reconcile the need for high comprehensiveness and temporal resolution with the key requirement of monitoring biological reproducibility.

DNA damage response: multilevel proteomics gains momentum.

In this issue of Molecular Cell, Beli et al. (2012) introduce a multilevel proteomics approach for parallel quantification of protein phosphorylation, acetylation, and abundance and apply this to the complex signaling network of the DNA damage response.

Global analysis of phosphoproteome regulation by the Ser/Thr phosphatase Ppt1 in Saccharomyces cerevisiae.

Even though protein phosphatases are key regulators of signal transduction, their cellular mechanisms of action are poorly understood. Here, we undertook a large-scale proteomics survey to identify cellular protein targets of a serine/threonine phosphatase. We used SILAC-based quantitative MS to measure differences in protein expression and phosphorylation upon ablation of the serine/threonine phosphatase Ppt1 in Saccharomyces cerevisiae.

Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation.

Hsp90 is an essential molecular chaperone in the eukaryotic cytosol. Its function is modulated by cochaperones and posttranslational modifications. Importantly, the phosphatase Ppt1 is a dedicated regulator of the Hsp90 chaperone system.

Combination of chemical genetics and phosphoproteomics for kinase signaling analysis enables confident identification of cellular downstream targets.

Delineation of phosphorylation-based signaling networks requires reliable data about the underlying cellular kinase-substrate interactions. We report a chemical genetics and quantitative phosphoproteomics approach that encompasses cellular kinase activation in combination with comparative replicate mass spectrometry analyses of cells expressing either inhibitor-sensitive or resistant kinase variant. We applied this workflow to Plk1 (Polo-like kinase 1) in mitotic cells and induced cellular Plk1 activity by wash-out of the bulky kinase inhibitor 3-MB-PP1, which targets a mutant kinase version with an enlarged catalytic pocket while not interfering with wild-type Plk1.

Dual phosphoproteomics and chemical proteomics analysis of erlotinib and gefitinib interference in acute myeloid leukemia cells.

Small molecule inhibitors of protein kinases have emerged as a major class of therapeutic agents for the treatment of hematological malignancies. Both in vitro studies and patient case reports suggest therapeutic potential of the clinical kinase inhibitors erlotinib and gefitinib in acute myeloid leukemia (AML). The drugs' cellular modes of action in AML warrant further investigation as their primary therapeutic target, the epidermal growth factor receptor, is not expressed.

Proteomics analysis of cellular imatinib targets and their candidate downstream effectors.

Inhibition of deregulated protein kinases by small molecule drugs has evolved into a major therapeutic strategy for the treatment of human malignancies. Knowledge about direct cellular targets of kinase-selective drugs and the identification of druggable downstream mediators of oncogenic signaling are relevant for both initial therapy selection and the nomination of alternative targets in case molecular resistance emerges. To address these issues, we performed a proof-of-concept proteomics study designed to monitor drug effects on the pharmacologically tractable subproteome isolated by affinity purification with immobilized, nonselective kinase inhibitors.

Glycoprotein capture and quantitative phosphoproteomics indicate coordinated regulation of cell migration upon lysophosphatidic acid stimulation.

The lipid mediator lysophosphatidic acid (LPA) is a serum component that regulates cellular functions such as proliferation, migration, and survival via specific G protein-coupled receptors. The underlying signaling mechanisms are still incompletely understood, including those that operate at the plasma membrane to modulate cell-cell and cell-matrix interactions in LPA-promoted cell migration. To explore LPA-evoked phosphoregulation with a focus on cell surface proteins, we combined glycoproteome enrichment by immobilized lectins with SILAC-based quantitative phosphoproteomics.

Quantitative analysis of kinase-proximal signaling in lipopolysaccharide-induced innate immune response.

The innate immune system senses invariant microbial components via toll-like receptors (TLRs) to elicit a host defense program against invading pathogens. Lipopolysaccharide (LPS), a constituent of Gram-negative bacteria, is recognized by TLR4 and triggers protein kinase signaling to orchestrate immune responses such as inflammatory cytokine production. To analyze kinase-proximal signaling in murine macrophages, we performed prefractionation experiments with immobilized kinase inhibitors to enrich for protein kinases and their interaction partners.

Quantitative site-specific phosphorylation dynamics of human protein kinases during mitotic progression.

Reversible protein phosphorylation is a key regulatory mechanism of mitotic progression. Importantly, protein kinases themselves are also regulated by phosphorylation-dephosphorylation processes; hence, phosphorylation dynamics of kinases hold a wealth of information about phosphorylation networks. Here, we investigated the site-specific phosphorylation dynamics of human kinases during mitosis using synchronization of HeLa suspension cells, kinase enrichment, and high resolution mass spectrometry.

An integrated phosphoproteomics work flow reveals extensive network regulation in early lysophosphatidic acid signaling.

Lysophosphatidic acid (LPA) induces a variety of cellular signaling pathways through the activation of its cognate G protein-coupled receptors. To investigate early LPA responses and assess the contribution of epidermal growth factor (EGF) receptor transactivation in LPA signaling, we performed phosphoproteomics analyses of both total cell lysate and protein kinase-enriched fractions as complementary strategies to monitor phosphorylation changes in A498 kidney carcinoma cells. Our integrated work flow enabled the identification and quantification of more than 5,300 phosphorylation sites of which 224 were consistently regulated by LPA.

Proteomics strategy for quantitative protein interaction profiling in cell extracts.

We report a proteomics strategy to both identify and quantify cellular target protein interactions with externally introduced ligands. We determined dissociation constants for target proteins interacting with the ligand of interest by combining quantitative mass spectrometry with a defined set of affinity purification experiments. We demonstrate the general utility of this methodology in interaction studies involving small-molecule kinase inhibitors, a tyrosine-phosphorylated peptide and an antibody as affinity ligands.

Global effects of kinase inhibitors on signaling networks revealed by quantitative phosphoproteomics.

Aberrant signaling causes many diseases, and manipulating signaling pathways with kinase inhibitors has emerged as a promising area of drug research. Most kinase inhibitors target the conserved ATP-binding pocket; therefore specificity is a major concern. Proteomics has previously been used to identify the direct targets of kinase inhibitors upon affinity purification from cellular extracts.