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). Our in-depth co-culture and in vivo analyses cover more than 10,000 cancer cell-derived proteins and reveal systematic differences between molecular PDAC subtypes. Secreted proteins, such as chemokines and EMT-promoting matrisome proteins, associated with distinct macrophage polarization and tumor stromal composition, differentiate classical and mesenchymal PDAC. Intriguingly, more than 1,600 cancer cell-derived proteins including cytokines and pre-metastatic niche formation-associated factors in mouse serum reflect tumor activity in circulation. Our findings highlight how cell-selective proteomics can accelerate the discovery of diagnostic markers and therapeutic targets in cancer.

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. Upon inhibition of the oncology target USP7, we simultaneously record ubiquitination and consequent changes in abundance of more than 8,000 proteins at high temporal resolution. While ubiquitination of hundreds of proteins increases within minutes of USP7 inhibition, we find that only a small fraction of those are ever degraded, thereby dissecting the scope of USP7 action. Our method enables rapid mode-of-action profiling of candidate drugs targeting DUBs or ubiquitin ligases at high precision and throughput.

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. Here, we report on systematic siRNA-based screening for modulators of the capacity of SMN to condense in Cajal bodies and identify mTOR and ribosomal protein S6 kinase ß-1 as key regulators. Proteomic analysis reveals TOR-dependent phosphorylations in SMN complex subunits. Using stably expressed or optogenetically controlled phospho mutants, we demonstrate that serine 49 and 63 phosphorylation of human SMN controls the capacity of the complex to condense in Cajal bodies via liquid-liquid phase separation. Our findings link SMN complex condensation and UsnRNP biogenesis to cellular energy levels and suggest modulation of TOR signaling as a rational concept for therapy of the SMN-linked neuromuscular disorder spinal muscular atrophy.

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. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex's conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.

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. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies.

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. On average, close to 4000 distinct proteins were identified and quantified per cell line. These included many known targets and diagnostic markers, indicating that our proteome expression data represents a useful resource for NSCLC pre-clinical research. To assess proteome diversity within the NSCLC cell line panel, we performed hierarchical clustering and principal component analysis of proteome expression data. Our results indicate that general proteome diversity among NSCLC cell lines supersedes potential effects common to K-Ras or epidermal growth factor receptor (EGFR) oncoprotein expression. However, we observed partial segregation of EGFR or KRAS mutant cell lines for certain principal components, which reflected biological differences according to gene ontology enrichment analyses. Moreover, statistical analysis revealed several proteins that were significantly overexpressed in KRAS or EGFR mutant cell lines.

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). RESULTS: Single-shot experiments identified on average about 5000 proteins in the label-free compared to about 3500 in super-SILAC experiments. Label-free quantification was slightly less precise than super-SILAC in replicate measurements, verifying previous results obtained for lower proteome coverage. Due to the higher number of quantified proteins, more significant differences were detected in label-free cell line comparisons, whereas a higher percentage of quantified proteins was identified as differentially expressed in super-SILAC experiments. Additional label-free replicate analyses effectively compensated for lower precision of quantification. Finally, peptide fractionation by high pH reversed-phase chromatography prior to LC/MS analysis further increased the robustness and precision of label-free quantification in conjunction with higher proteome coverage. CONCLUSIONS: Our results benchmark and highlight the utility of label-free proteome quantification for applications such as target and biomarker discovery on state-of-the-art UHPLC/MS workflows.

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. Thus, critical information is provided to promote new insights into mechanisms of kinase signaling and their relevance for kinase inhibitor drug discovery.

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. The SMN complex is subject to extensive phosphorylation. Detailed understanding of SMN complex regulation necessitates a comprehensive analysis of these post-translational modifications. Here, we report on the first comprehensive phosphoproteome analysis of the intact human SMN complex, which identify 48 serine/threonine phosphosites in the complex. We find that 7 out of 9 SMN components of the intact complex are phosphoproteins and confidently place 29 phosphorylation sites, 12 of them in SMN itself. By the generation of multi non-phosphorylatable or phosphomimetic variants of SMN, respectively, we address to which extent phosphorylation regulates SMN complex function and localization. Both phosphomimetic and non-phosphorylatable variants assemble into intact SMN complexes and can compensate the loss of endogenous SMN in snRNP assembly at least to some extent. However, they partially or completely fail to target to nuclear Cajal bodies. Moreover, using a mutant of SMN, which cannot be phosphorylated on previously reported tyrosine residues, we provide first evidence that this PTM regulates SMN localization and nuclear accumulation. Our data suggest complex regulatory cues mediated by phosphorylation of serine/threonine and tyrosine residues, which control the subcellular localization of the SMN complex and its accumulation in nuclear CB.

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. Both enabled high phosphoproteome coverage in H3122 lung cancer cells. Replicate quantifications by mTRAQ identified almost as many significant phosphorylation changes upon treatment with ALK kinase inhibitor crizotinib as found by SILAC quantification. Overall, mTRAQ was slightly less precise than SILAC as evident from a somewhat higher variance of replicate phosphosite ratios. Direct comparison of SILAC- and mTRAQ-quantified phosphosites revealed that the majority of changes were detected by either quantification techniques, but also highlighted the aspect of false negative identifications in quantitative proteomics applications. Further inspection of crizotinib-regulated phosphorylation changes unveiled interference with multiple antioncogenic mechanisms downstream of ALK fusion kinase in H3122 cells. In conclusion, our results demonstrate a strong analytical performance of the Q Exactive in global phosphoproteomics, and establish mTRAQ quantification as a useful alternative to metabolic isotope labeling.

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. We treated SILAC-labeled SCC-9 cells with the seven transmembrane receptor ligand lysophosphatidic acid (LPA) and identified more than 17 000 phosphorylation sites. Filtering for biological replicate quantification yielded five-time point profiles for 6292 site-specific phosphorylations, which we analyzed for statistically significant regulation. Notably, about 30% of these sites changed significantly upon LPA stimulation, indicating extensive phosphoproteome regulation in response to this growth factor. Analysis of time series data identified distinct temporal profiles for different kinase substrate motifs, likely reflecting temporal orchestration of cellular kinase activities. Our data further indicated coordinated regulation of biological processes and phosphoprotein networks upon LPA stimulation. Finally, we detected regulation of functionally characterized phosphorylation sites not yet implicated in LPA signaling, which may foster a better understanding how LPA regulates cellular physiology on the molecular level.

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.

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. Little is known about Ppt1-dependent phosphorylation sites and how these affect Hsp90 activity. Here, we identified the major phosphorylation sites of yeast Hsp90 in its middle or the C-terminal domain and determined the subset regulated by Ppt1. In general, phosphorylation decelerates the Hsp90 machinery, reduces chaperone function in vivo, sensitizes yeast cells to Hsp90 inhibition and affects DNA repair processes. Modification of one particular site (S485) is lethal, whereas others modulate Hsp90 activity via distinct mechanisms affecting the ATPase activity, cochaperone binding and manipulating conformational transitions in Hsp90. Our mechanistic analysis reveals that phosphorylation of Hsp90 permits a regulation of the conformational cycle at distinct steps by targeting switch points for the communication of remote regions within Hsp90.

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. Phosphopeptide fractionation by strong cation exchange chromatography combined with immobilized metal affinity chromatography (IMAC) enrichment enabled quantification of more than 8000 distinct phosphorylation sites in Ppt1 wild-type versus Ppt1-deficient yeast cells. We further quantified the relative expression of 1897 yeast proteins and detected no major protein changes accompanying Ppt1 deficiency. Notably, we found 33 phosphorylation sites to be significantly and reproducibly up-regulated while no phosphorylation events were repressed in cells lacking Ppt1. Ppt1 acted on its cellular target proteins in a sequence- and site-specific fashion. Several of the regulated phosphoproteins were involved in the response to heat stress in agreement with known Ppt1 functions. Additionally, biosynthetic enzymes were particularly prominent among Ppt1-regulated phosphoproteins, pointing to unappreciated roles of Ppt1 in the control of various metabolic functions. These results demonstrate the utility of large-scale and quantitative phosphoproteomics to identify cellular sites of serine/threonine phosphatase action in an unbiased manner.

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. We therefore performed SILAC-based quantitative mass spectrometry analyses to a depth of 10,975 distinct phosphorylation sites to characterize the phosphoproteome of KG1 AML cells and its regulation upon erlotinib and gefitinib treatment. Less than 50 site-specific phosphorylations changed significantly, indicating rather specific interference with AML cell signaling. Many drug-induced changes occurred within a network of tyrosine phosphorylated proteins that included Src family kinases (SFKs) and the tyrosine kinases Btk and Syk. We further performed quantitative chemical proteomics in KG1 cell extracts and identified SFKs and Btk as direct cellular targets of both erlotinib and gefitinib. Taken together, our data suggest that cellular perturbation of SFKs and/or Btk translates into rather specific signal transduction inhibition, which in turn contributes to the antileukemic activity of erlotinib and gefitinib in AML.

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. We quantified more than 20,000 distinct phosphorylation sites by SILAC, approximately half of which were measured in at least two independent experiments in cells expressing mutant and wild-type Plk1. Based on replicate phosphorylation site quantifications in both mutant and wild-type Plk1 cells, our chemical genetic proteomics concept enabled stringent comparative statistics by significance analysis of microarrays, which unveiled more than 350 cellular downstream targets of Plk1 validated by full concordance of both statistical and experimental data. Our data point to hitherto poorly characterized aspects in Plk1-controlled mitotic progression and provide a largely extended resource for functional studies. We anticipate the described strategies to be of general utility for systematic and confident identification of cellular protein kinase substrates.

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. We applied this strategy to chronic myeloid leukemia cells that express the transforming Bcr-Abl fusion kinase. We used SILAC to measure how cellular treatment with the Bcr-Abl inhibitor imatinib affects protein binding to a generic kinase inhibitor resin and further quantified site-specific phosphorylations on resin-retained proteins. Our integrated approach indicated additional imatinib target candidates, such as flavine adenine dinucleotide synthetase, as well as repressed phosphorylation events on downstream effectors not yet implicated in imatinib-regulated signaling. These included activity-regulating phosphorylations on the kinases Btk, Fer, and focal adhesion kinase, which may qualify them as alternative target candidates in Bcr-Abl-driven oncogenesis. Our approach is rather generic and may have various applications in kinase drug discovery.

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. We performed biological replicate analyses in SCC-9 squamous cell carcinoma cells and repeatedly quantified the effect of 1.5- and 5-min LPA treatment on more than 700 distinct phosphorylations in lectin-purified proteins. We detected many regulated phosphorylation events on various types of plasma membrane proteins such as cell adhesion molecules constituting adherens junctions, desmosomes, and hemidesmosomes. Several of these LPA-regulated phosphorylation sites have been characterized in a biological context other than G protein-coupled receptor signaling, and the transfer of this functional information suggests coordinated and multifactorial cell adhesion control in LPA-induced cell migration. Additionally, we identified LPA-mediated activation loop phosphorylation of the serine/threonine kinase Wnk1 and verified a role of Wnk1 for LPA-induced cell migration in knock-down experiments. In conclusion, the glycoproteome phosphoproteomics strategy described here sheds light on incompletely understood mechanisms in LPA-induced cell migratory behavior.

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. In conjunction with SILAC-based quantitative mass spectrometry and phosphopeptide enrichment, we recorded five time point profiles for more than 850 distinct phosphorylation events on protein kinases and copurifying factors. More than 15% exhibited significant changes and many of those mapped to LPS-regulated kinase networks. We identified many unreported TLR signaling events including LPS-triggered phosphorylations of Akt substrates, which point to previously unknown molecular mechanisms in innate immune response. We further detected extensive phosphoregulation of TANK-binding kinase 1, inhibitor of nuclear factor-kappaB kinase epsilon and their associating scaffolding factors, and none of these events were known despite the key roles of these proteins in LPS signaling. Thus, our data expands previous knowledge for functional analyses of innate immune response.