A cross-species spatiotemporal proteomic analysis identifies UBE3A-dependent signaling pathways and targets.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of neuronal E3 ligase UBE3A. Restoring UBE3A levels is a potential disease-modifying therapy for AS and has recently entered clinical trials. There is paucity of data regarding the molecular changes downstream of UBE3A hampering elucidation of disease therapeutics and biomarkers. Notably, UBE3A plays an important role in the nucleus but its targets have yet to be elucidated. Using proteomics, we assessed changes during postnatal cortical development in an AS mouse model. Pathway analysis revealed dysregulation of proteasomal and tRNA synthetase pathways at all postnatal brain developmental stages, while synaptic proteins were altered in adults. We confirmed pathway alterations in an adult AS rat model across multiple brain regions and highlighted region-specific differences. UBE3A reinstatement in AS model mice resulted in near complete and partial rescue of the proteome alterations in adolescence and adults, respectively, supporting the notion that restoration of UBE3A expression provides a promising therapeutic option. We show that the nuclear enriched transketolase (TKT), one of the most abundantly altered proteins, is a novel direct UBE3A substrate and is elevated in the neuronal nucleus of rat brains and human iPSC-derived neurons. Taken together, our study provides a comprehensive map of UBE3A-driven proteome remodeling in AS across development and species, and corroborates an early UBE3A reinstatement as a viable therapeutic option. To support future disease and biomarker research, we present an accessible large-scale multi-species proteomic resource for the AS community ( https://www.angelman-proteome-project.org/ ).

The Perseus computational platform for comprehensive analysis of (prote)omics data.

A main bottleneck in proteomics is the downstream biological analysis of highly multivariate quantitative protein abundance data generated using mass-spectrometry-based analysis. We developed the Perseus software platform (http://www.perseus-framework.org) to support biological and biomedical researchers in interpreting protein quantification, interaction and post-translational modification data. Perseus contains a comprehensive portfolio of statistical tools for high-dimensional omics data analysis covering normalization, pattern recognition, time-series analysis, cross-omics comparisons and multiple-hypothesis testing. A machine learning module supports the classification and validation of patient groups for diagnosis and prognosis, and it also detects predictive protein signatures. Central to Perseus is a user-friendly, interactive workflow environment that provides complete documentation of computational methods used in a publication. All activities in Perseus are realized as plugins, and users can extend the software by programming their own, which can be shared through a plugin store. We anticipate that Perseus's arsenal of algorithms and its intuitive usability will empower interdisciplinary analysis of complex large data sets.

Proteomics reveals the effects of sustained weight loss on the human plasma proteome.

Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill-defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed by a year of weight maintenance. Using mass spectrometry-based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual-specific protein levels with wide-ranging effects of losing weight on the plasma proteome reflected in 93 significantly affected proteins. The adipocyte-secreted SERPINF1 and apolipoprotein APOF1 were most significantly regulated with fold changes of -16% and +37%, respectively (P < 10-13), and the entire apolipoprotein family showed characteristic differential regulation. Clinical laboratory parameters are reflected in the plasma proteome, and eight plasma proteins correlated better with insulin resistance than the known marker adiponectin. Nearly all study participants benefited from weight loss regarding a ten-protein inflammation panel defined from the proteomics data. We conclude that plasma proteome profiling broadly evaluates and monitors intervention in metabolic diseases.

Machine Learning-based Classification of Diffuse Large B-cell Lymphoma Patients by Their Protein Expression Profiles.

Characterization of tumors at the molecular level has improved our knowledge of cancer causation and progression. Proteomic analysis of their signaling pathways promises to enhance our understanding of cancer aberrations at the functional level, but this requires accurate and robust tools. Here, we develop a state of the art quantitative mass spectrometric pipeline to characterize formalin-fixed paraffin-embedded tissues of patients with closely related subtypes of diffuse large B-cell lymphoma. We combined a super-SILAC approach with label-free quantification (hybrid LFQ) to address situations where the protein is absent in the super-SILAC standard but present in the patient samples. Shotgun proteomic analysis on a quadrupole Orbitrap quantified almost 9,000 tumor proteins in 20 patients. The quantitative accuracy of our approach allowed the segregation of diffuse large B-cell lymphoma patients according to their cell of origin using both their global protein expression patterns and the 55-protein signature obtained previously from patient-derived cell lines (Deeb, S. J., D'Souza, R. C., Cox, J., Schmidt-Supprian, M., and Mann, M. (2012) Mol. Cell. Proteomics 11, 77-89). Expression levels of individual segregation-driving proteins as well as categories such as extracellular matrix proteins behaved consistently with known trends between the subtypes. We used machine learning (support vector machines) to extract candidate proteins with the highest segregating power. A panel of four proteins (PALD1, MME, TNFAIP8, and TBC1D4) is predicted to classify patients with low error rates. Highly ranked proteins from the support vector analysis revealed differential expression of core signaling molecules between the subtypes, elucidating aspects of their pathobiology.

Visualization of LC-MS/MS proteomics data in MaxQuant.

Modern software platforms enable the analysis of shotgun proteomics data in an automated fashion resulting in high quality identification and quantification results. Additional understanding of the underlying data can be gained with the help of advanced visualization tools that allow for easy navigation through large LC-MS/MS datasets potentially consisting of terabytes of raw data. The updated MaxQuant version has a map navigation component that steers the users through mass and retention time-dependent mass spectrometric signals. It can be used to monitor a peptide feature used in label-free quantification over many LC-MS runs and visualize it with advanced 3D graphic models. An expert annotation system aids the interpretation of the MS/MS spectra used for the identification of these peptide features.

Phosphorylation variation during the cell cycle scales with structural propensities of proteins.

Phosphorylation at specific residues can activate a protein, lead to its localization to particular compartments, be a trigger for protein degradation and fulfill many other biological functions. Protein phosphorylation is increasingly being studied at a large scale and in a quantitative manner that includes a temporal dimension. By contrast, structural properties of identified phosphorylation sites have so far been investigated in a static, non-quantitative way. Here we combine for the first time dynamic properties of the phosphoproteome with protein structural features. At six time points of the cell division cycle we investigate how the variation of the amount of phosphorylation correlates with the protein structure in the vicinity of the modified site. We find two distinct phosphorylation site groups: intrinsically disordered regions tend to contain sites with dynamically varying levels, whereas regions with predominantly regular secondary structures retain more constant phosphorylation levels. The two groups show preferences for different amino acids in their kinase recognition motifs - proline and other disorder-associated residues are enriched in the former group and charged residues in the latter. Furthermore, these preferences scale with the degree of disorderedness, from regular to irregular and to disordered structures. Our results suggest that the structural organization of the region in which a phosphorylation site resides may serve as an additional control mechanism. They also imply that phosphorylation sites are associated with different time scales that serve different functional needs.

RWD-derived response in multiple myeloma.

Real-world data (RWD) are important for understanding the treatment course and response patterns of patients with multiple myeloma. This exploratory pilot study establishes a way to reliably assess response from incomplete laboratory measurements captured in RWD. A rule-based algorithm, adapted from International Myeloma Working Group response criteria, was used to derive response using RWD. This derived response (dR) algorithm was assessed using data from the phase III BELLINI trial, comparing the number of responders and non-responders assigned by independent review committee (IRC) versus the dR algorithm. To simulate a real-world scenario with missing data, a sensitivity analysis was conducted whereby available laboratory measurements in the dataset were artificially reduced. Associations between dR and overall survival were evaluated at 1) individual level and 2) treatment level in a real-world patient cohort obtained from a nationwide electronic health record-derived de-identified database. The algorithm's assignment of responders was highly concordant with that of the IRC (Cohen's Kappa 0.83) using the BELLINI data. The dR replicated the differences in overall response rate between the intervention and placebo arms reported in the trial (odds ratio 2.1 vs. 2.3 for IRC vs. dR assessment, respectively). Simulation of missing data in the sensitivity analysis (-50% of available laboratory measurements and -75% of urine monoclonal protein measurements) resulted in a minor reduction in the algorithm's accuracy (Cohen's Kappa 0.75). In the RWD cohort, dR was significantly associated with overall survival at all landmark times (hazard ratios 0.80-0.81, p<0.001) at the individual level, while the overall association was R2 = 0.67 (p<0.001) at the treatment level. This exploratory pilot study demonstrates the feasibility of deriving accurate response from RWD. With further confirmation in independent cohorts, the dR has the potential to be used as an endpoint in real-world studies and as a comparator in single-arm clinical trials.

Increased G3BP2-Tau interaction in tauopathies is a natural defense against Tau aggregation.

Many RNA-binding proteins (RBPs), particularly those associated with RNA granules, promote pathological protein aggregation in neurodegenerative diseases. Here, we demonstrate that G3BP2, a core component of stress granules, directly interacts with Tau and inhibits Tau aggregation. In the human brain, the interaction of G3BP2 and Tau is dramatically increased in multiple tauopathies, and it is independent of neurofibrillary tangle (NFT) formation in Alzheimer's disease (AD). Surprisingly, Tau pathology is significantly elevated upon loss of G3BP2 in human neurons and brain organoids. Moreover, we found that G3BP2 masks the microtubule-binding region (MTBR) of Tau, thereby inhibiting Tau aggregation. Our study defines a novel role for RBPs as a line of defense against Tau aggregation in tauopathies.

Targeted suppression of mTORC2 reduces seizures across models of epilepsy.

Epilepsy is a neurological disorder that poses a major threat to public health. Hyperactivation of mTOR complex 1 (mTORC1) is believed to lead to abnormal network rhythmicity associated with epilepsy, and its inhibition is proposed to provide some therapeutic benefit. However, mTOR complex 2 (mTORC2) is also activated in the epileptic brain, and little is known about its role in seizures. Here we discover that genetic deletion of mTORC2 from forebrain neurons is protective against kainic acid-induced behavioral and EEG seizures. Furthermore, inhibition of mTORC2 with a specific antisense oligonucleotide robustly suppresses seizures in several pharmacological and genetic mouse models of epilepsy. Finally, we identify a target of mTORC2, Nav1.2, which has been implicated in epilepsy and neuronal excitability. Our findings, which are generalizable to several models of human seizures, raise the possibility that inhibition of mTORC2 may serve as a broader therapeutic strategy against epilepsy.

DUSP4 protects BRAF- and NRAS-mutant melanoma from oncogene overdose through modulation of MITF.

MAPK inhibitors (MAPKi) remain an important component of the standard of care for metastatic melanoma. However, acquired resistance to these drugs limits their therapeutic benefit. Tumor cells can become refractory to MAPKi by reactivation of ERK. When this happens, tumors often become sensitive to drug withdrawal. This drug addiction phenotype results from the hyperactivation of the oncogenic pathway, a phenomenon commonly referred to as oncogene overdose. Several feedback mechanisms are involved in regulating ERK signaling. However, the genes that serve as gatekeepers of oncogene overdose in mutant melanoma remain unknown. Here, we demonstrate that depletion of the ERK phosphatase, DUSP4, leads to toxic levels of MAPK activation in both drug-naive and drug-resistant mutant melanoma cells. Importantly, ERK hyperactivation is associated with down-regulation of lineage-defining genes including MITF Our results offer an alternative therapeutic strategy to treat mutant melanoma patients with acquired MAPKi resistance and those unable to tolerate MAPKi.

SILAC-based quantitative proteomics using mass spectrometry quantifies endoplasmic reticulum stress in whole HeLa cells.

The unfolded protein response (UPR) involves extensive proteome remodeling in many cellular compartments. To date, a comprehensive analysis of the UPR has not been possible because of technological limitations. Here, we employ stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics to quantify the response of over 6200 proteins to increasing concentrations of tunicamycin in HeLa cells. We further compare the effects of tunicamycin (5 µg/ml) to those of thapsigargin (1 µM) and DTT (2 mM), both activating the UPR through different mechanisms. This systematic quantification of the proteome-wide expression changes that follow proteostatic stress is a resource for the scientific community, enabling the discovery of novel players involved in the pathophysiology of the broad range of disorders linked to proteostasis. We identified increased expression in 38 proteins not previously linked to the UPR, of which 15 likely remediate ER stress, and the remainder may contribute to pathological outcomes. Unexpectedly, there are few strongly downregulated proteins, despite expression of the pro-apoptotic transcription factor CHOP, suggesting that IRE1-dependent mRNA decay (RIDD) has a limited contribution to ER stress-mediated cell death in our system.

Perseus: A Bioinformatics Platform for Integrative Analysis of Proteomics Data in Cancer Research.

Mass spectrometry-based proteomics is a continuously growing field marked by technological and methodological improvements. Cancer proteomics is aimed at pursuing goals such as accurate diagnosis, patient stratification, and biomarker discovery, relying on the richness of information of quantitative proteome profiles. Translating these high-dimensional data into biological findings of clinical importance necessitates the use of robust and powerful computational tools and methods. In this chapter, we provide a detailed description of standard analysis steps for a clinical proteomics dataset performed in Perseus, a software for functional analysis of large-scale quantitative omics data.

Multiplexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice.

We describe a protocol for multiplexed proteomic analysis using neutron-encoded (NeuCode) stable isotope labeling of amino acids in cells (SILAC) or mice (SILAM). This method currently enables simultaneous comparison of up to nine treatment and control proteomes. Another important advantage over traditional SILAC/SILAM is that shorter labeling times are required. Exploiting the small mass differences that correspond to subtle differences in the neutron-binding energies of different isotopes, the amino acids used in NeuCode SILAC/SILAM differ in mass by just a few milliDaltons. Isotopologs of lysine are introduced into cells or mammals, via the culture medium or diet, respectively, to metabolically label the proteome. Labeling time is ∼2 weeks for cultured cells and 3-4 weeks for mammals. The proteins are then extracted, relevant samples are combined, and these are enzymatically digested with lysyl endopeptidase (Lys-C). The resultant peptides are chromatographically separated and then mass analyzed. During mass spectrometry (MS) data acquisition, high-resolution MS1 spectra (≥240,000 resolving power at m/z = 400) reveal the embedded isotopic signatures, enabling relative quantification, while tandem mass spectra, collected at lower resolutions, provide peptide identities. Both types of spectra are processed using NeuCode-enabled MaxQuant software. In total, the approximate completion time for the protocol is 3-5 weeks.

The Proteome of Prostate Cancer Bone Metastasis Reveals Heterogeneity with Prognostic Implications.

Purpose: Bone is the most predominant site of distant metastasis in prostate cancer, and patients have limited therapeutic options at this stage.Experimental Design: We performed a system-wide quantitative proteomic analysis of bone metastatic prostate tumors from 22 patients operated to relieve spinal cord compression. At the time of surgery, most patients had relapsed after androgen-deprivation therapy, while 5 were previously untreated. An extended cohort of prostate cancer bone metastases (n = 65) was used for immunohistochemical validation.Results: On average, 5,067 proteins were identified and quantified per tumor. Compared with primary tumors (n = 26), bone metastases were more heterogeneous and showed increased levels of proteins involved in cell-cycle progression, DNA damage response, RNA processing, and fatty acid ß-oxidation; and reduced levels of proteins were related to cell adhesion and carbohydrate metabolism. Within bone metastases, we identified two phenotypic subgroups: BM1, expressing higher levels of AR canonical targets, and mitochondrial and Golgi apparatus resident proteins; and BM2, with increased expression of proliferation and DNA repair-related proteins. The two subgroups, validated by the inverse correlation between MCM3 and prostate specific antigen immunoreactivity, were related to disease prognosis, suggesting that this molecular heterogeneity should be considered when developing personalized therapies.Conclusions: This work is the first system-wide quantitative characterization of the proteome of prostate cancer bone metastases and a valuable resource for understanding the etiology of prostate cancer progression. Clin Cancer Res; 24(21); 5433-44. ©2018 AACR.

A Mass Spectrometry-Based Approach for Mapping Protein Subcellular Localization Reveals the Spatial Proteome of Mouse Primary Neurons.

We previously developed a mass spectrometry-based method, dynamic organellar maps, for the determination of protein subcellular localization and identification of translocation events in comparative experiments. The use of metabolic labeling for quantification (stable isotope labeling by amino acids in cell culture [SILAC]) renders the method best suited to cells grown in culture. Here, we have adapted the workflow to both label-free quantification (LFQ) and chemical labeling/multiplexing strategies (tandem mass tagging [TMT]). Both methods are highly effective for the generation of organellar maps and capture of protein translocations. Furthermore, application of label-free organellar mapping to acutely isolated mouse primary neurons provided subcellular localization and copy-number information for over 8,000 proteins, allowing a detailed analysis of organellar organization. Our study extends the scope of dynamic organellar maps to any cell type or tissue and also to high-throughput screening.

The MaxQuant computational platform for mass spectrometry-based shotgun proteomics.

MaxQuant is one of the most frequently used platforms for mass-spectrometry (MS)-based proteomics data analysis. Since its first release in 2008, it has grown substantially in functionality and can be used in conjunction with more MS platforms. Here we present an updated protocol covering the most important basic computational workflows, including those designed for quantitative label-free proteomics, MS1-level labeling and isobaric labeling techniques. This protocol presents a complete description of the parameters used in MaxQuant, as well as of the configuration options of its integrated search engine, Andromeda. This protocol update describes an adaptation of an existing protocol that substantially modifies the technique. Important concepts of shotgun proteomics and their implementation in MaxQuant are briefly reviewed, including different quantification strategies and the control of false-discovery rates (FDRs), as well as the analysis of post-translational modifications (PTMs). The MaxQuant output tables, which contain information about quantification of proteins and PTMs, are explained in detail. Furthermore, we provide a short version of the workflow that is applicable to data sets with simple and standard experimental designs. The MaxQuant algorithms are efficiently parallelized on multiple processors and scale well from desktop computers to servers with many cores. The software is written in C# and is freely available at http://www.maxquant.org.

Global, quantitative and dynamic mapping of protein subcellular localization.

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology.

Proteomic maps of breast cancer subtypes.

Systems-wide profiling of breast cancer has almost always entailed RNA and DNA analysis by microarray and sequencing techniques. Marked developments in proteomic technologies now enable very deep profiling of clinical samples, with high identification and quantification accuracy. We analysed 40 oestrogen receptor positive (luminal), Her2 positive and triple negative breast tumours and reached a quantitative depth of >10,000 proteins. These proteomic profiles identified functional differences between breast cancer subtypes, related to energy metabolism, cell growth, mRNA translation and cell-cell communication. Furthermore, we derived a signature of 19 proteins, which differ between the breast cancer subtypes, through support vector machine (SVM)-based classification and feature selection. Remarkably, only three proteins of the signature were associated with gene copy number variations and eleven were also reflected on the mRNA level. These breast cancer features revealed by our work provide novel insights that may ultimately translate to development of subtype-specific therapeutics.

The Proteome of Primary Prostate Cancer.

BACKGROUND: Clinical management of the prostate needs improved prognostic tests and treatment strategies. Because proteins are the ultimate effectors of most cellular reactions, are targets for drug actions and constitute potential biomarkers; a quantitative systemic overview of the proteome changes occurring during prostate cancer (PCa) initiation and progression can result in clinically relevant discoveries. OBJECTIVES: To study cellular processes altered in PCa using system-wide quantitative analysis of changes in protein expression in clinical samples and to identify prognostic biomarkers for disease aggressiveness. DESIGN, SETTING, AND PARTICIPANTS: Mass spectrometry was used for genome-scale quantitative proteomic profiling of 28 prostate tumors (Gleason score 6-9) and neighboring nonmalignant tissue in eight cases, obtained from formalin-fixed paraffin-embedded prostatectomy samples. Two independent cohorts of PCa patients (summing 752 cases) managed by expectancy were used for immunohistochemical evaluation of proneuropeptide-Y (pro-NPY) as a prognostic biomarker. RESULTS AND LIMITATIONS: Over 9000 proteins were identified as expressed in the human prostate. Tumor tissue exhibited elevated expression of proteins involved in multiple anabolic processes including fatty acid and protein synthesis, ribosomal biogenesis and protein secretion but no overt evidence of increased proliferation was observed. Tumors also showed increased levels of mitochondrial proteins, which was associated with elevated oxidative phosphorylation capacity measured in situ. Molecular analysis indicated that some of the proteins overexpressed in tumors, such as carnitine palmitoyltransferase 2 (CPT2, fatty acid transporter), coatomer protein complex, subunit alpha (COPA, vesicle secretion), and mitogen- and stress-activated protein kinase 1 and 2 (MSK1/2, protein kinase) regulate the proliferation of PCa cells. Additionally, pro-NPY was found overexpressed in PCa (5-fold, p<0.05), but largely absent in other solid tumor types. Pro-NPY expression, alone or in combination with the ERG status of the tumor, was associated with an increased risk of PCa specific mortality, especially in patients with Gleason score ≤ 7 tumors. CONCLUSIONS: This study represents the first system-wide quantitative analysis of proteome changes associated to localized prostate cancer and as such constitutes a valuable resource for understanding the complex metabolic changes occurring in this disease. We also demonstrated that pro-NPY, a protein that showed differential expression between high and low risk tumors in our proteomic analysis, is also a PCa specific prognostic biomarker associated with increased risk for disease specific death in patients carrying low risk tumors. PATIENT SUMMARY: The identification of proteins whose expression change in prostate cancer provides novel mechanistic information related to the disease etiology. We hope that future studies will prove the value of this proteome dataset for development of novel therapies and biomarkers.

Cell type- and brain region-resolved mouse brain proteome.

Brain transcriptome and connectome maps are being generated, but an equivalent effort on the proteome is currently lacking. We performed high-resolution mass spectrometry-based proteomics for in-depth analysis of the mouse brain and its major brain regions and cell types. Comparisons of the 12,934 identified proteins in oligodendrocytes, astrocytes, microglia and cortical neurons with deep sequencing data of the transcriptome indicated deep coverage of the proteome. Cell type-specific proteins defined as tenfold more abundant than average expression represented about a tenth of the proteome, with an overrepresentation of cell surface proteins. To demonstrate the utility of our resource, we focused on this class of proteins and identified Lsamp, an adhesion molecule of the IgLON family, as a negative regulator of myelination. Our findings provide a framework for a system-level understanding of cell-type diversity in the CNS and serves as a rich resource for analyses of brain development and function.