Translation and evaluation of a pre-clinical 5-protein response prediction signature in a breast cancer phase Ib clinical trial.

Human protein biomarker discovery relies heavily on pre-clinical models, in particular established cell lines and patient-derived xenografts, but confirmation studies in primary tissue are essential to demonstrate clinical relevance. We describe in this study the process that was followed to clinically translate a 5-protein response signature predictive for the activity of an anti-HER3 monoclonal antibody (lumretuzumab) originally measured in fresh frozen xenograft tissue. We detail the development, qualification, and validation of the multiplexed targeted mass spectrometry assay used to assess the signature performance in formalin-fixed, paraffin-embedded human clinical samples collected in a phase Ib trial designed to evaluate lumretuzumab in patients with metastatic breast cancer.

Compartment-resolved Proteomic Analysis of Mouse Aorta during Atherosclerotic Plaque Formation Reveals Osteoclast-specific Protein Expression.

Atherosclerosis leads to vascular lesions that involve major rearrangements of the vascular proteome, especially of the extracellular matrix (ECM). Using single aortas from ApoE knock out mice, we quantified formation of plaques by single-run, high-resolution mass spectrometry (MS)-based proteomics. To probe localization on a proteome-wide scale we employed quantitative detergent solubility profiling.

Phosphoproteome Analysis Reveals Differential Mode of Action of Sorafenib in Wildtype and Mutated FLT3 Acute Myeloid Leukemia (AML) Cells.

Constitutively activating internal tandem duplication (ITD) alterations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) are common in acute myeloid leukemia (AML) and classifies FLT3 as an attractive therapeutic target. So far, applications of FLT3 small molecule inhibitors have been investigated primarily in FLT3-ITD patients. Only recently, a prolonged event-free survival has been observed in AML patients who were treated with the multikinase inhibitor sorafenib in addition to standard therapy.

Phosphodiesterase 10A Inhibition Improves Cortico-Basal Ganglia Function in Huntington's Disease Models.

Huntington's disease (HD) symptoms are driven to a large extent by dysfunction of the basal ganglia circuitry. HD patients exhibit reduced striatal phoshodiesterase 10 (PDE10) levels. Using HD mouse models that exhibit reduced PDE10, we demonstrate the benefit of pharmacologic PDE10 inhibition to acutely correct basal ganglia circuitry deficits.

Integrated genomics and proteomics define huntingtin CAG length-dependent networks in mice.

To gain insight into how mutant huntingtin (mHtt) CAG repeat length modifies Huntington's disease (HD) pathogenesis, we profiled mRNA in over 600 brain and peripheral tissue samples from HD knock-in mice with increasing CAG repeat lengths. We found repeat length-dependent transcriptional signatures to be prominent in the striatum, less so in cortex, and minimal in the liver. Coexpression network analyses revealed 13 striatal and 5 cortical modules that correlated highly with CAG length and age, and that were preserved in HD models and sometimes in patients.

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.

Time- and compartment-resolved proteome profiling of the extracellular niche in lung injury and repair.

The extracellular matrix (ECM) is a key regulator of tissue morphogenesis and repair. However, its composition and architecture are not well characterized. Here, we monitor remodeling of the extracellular niche in tissue repair in the bleomycin-induced lung injury mouse model.

Pareto Optimization Identifies Diverse Set of Phosphorylation Signatures Predicting Response to Treatment with Dasatinib.

Multivariate biomarkers that can predict the effectiveness of targeted therapy in individual patients are highly desired. Previous biomarker discovery studies have largely focused on the identification of single biomarker signatures, aimed at maximizing prediction accuracy. Here, we present a different approach that identifies multiple biomarkers by simultaneously optimizing their predictive power, number of features, and proximity to the drug target in a protein-protein interaction network.

Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling.

Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells. Despite great advances in mass-spectrometry-based proteomics, the extent, localization, and site-specific stoichiometry of this posttranslational modification (PTM) are unknown. Here, we develop a stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line.

Identification of significant features by the Global Mean Rank test.

With the introduction of omics-technologies such as transcriptomics and proteomics, numerous methods for the reliable identification of significantly regulated features (genes, proteins, etc.) have been developed. Experimental practice requires these tests to successfully deal with conditions such as small numbers of replicates, missing values, non-normally distributed expression levels, and non-identical distributions of features.

Beyond the proteome: Mass Spectrometry Special Interest Group (MS-SIG) at ISMB/ECCB 2013.

wenzhong.xiao@mgh.harvard.

Delayed times to tissue fixation result in unpredictable global phosphoproteome changes.

Protein phosphorylation controls the activity of signal transduction pathways regulated by kinases and phosphatases. Little is known, however, about the impact of preanalytical factors, for example, delayed times to tissue fixation, on global phosphoprotein levels in tissues. The aim of this study was to characterize the potential effects of delayed tissue preservation (cold ischemia) on the levels of phosphoproteins using targeted and nontargeted proteomic approaches.

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.

A SILAC-based approach identifies substrates of caspase-dependent cleavage upon TRAIL-induced apoptosis.

The extracellular ligand-induced extrinsic pathway of apoptosis is executed via caspase protease cascades that activate downstream effectors by means of site-directed proteolysis. Here we identify proteome changes upon the induction of apoptosis by the cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a Jurkat T cell line. We detected caspase-dependent cleavage substrates by quantifying protein intensities before and after TRAIL induction in SDS gel slices.

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.

Global quantitative phosphoproteome analysis of human tumor xenografts treated with a CD44 antagonist.

The cell surface glycoprotein CD44 plays an important role in the development and progression of various tumor types. RG7356 is a humanized antibody targeting the constant region of CD44 that shows antitumor efficacy in mice implanted with CD44-expressing tumors such as MDA-MB-231 breast cancer cells. CD44 receptor seems to function as the main receptor for hyaluronic acid and osteopontin, serving as coreceptor for growth factor pathways like cMet, EGFR, HER-2, and VEGFR and by cytoskeletal modulation via ERM and Rho kinase signaling.

Phosphosignature predicts dasatinib response in non-small cell lung cancer.

Targeted drugs are less toxic than traditional chemotherapeutic therapies; however, the proportion of patients that benefit from these drugs is often smaller. A marker that confidently predicts patient response to a specific therapy would allow an individual therapy selection most likely to benefit the patient. Here, we used quantitative mass spectrometry to globally profile the basal phosphoproteome of a panel of non-small cell lung cancer cell lines.

Analysis of high accuracy, quantitative proteomics data in the MaxQB database.

MS-based proteomics generates rapidly increasing amounts of precise and quantitative information. Analysis of individual proteomic experiments has made great strides, but the crucial ability to compare and store information across different proteome measurements still presents many challenges. For example, it has been difficult to avoid contamination of databases with low quality peptide identifications, to control for the inflation in false positive identifications when combining data sets, and to integrate quantitative data.

Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins.

Deep proteomic analysis of mammalian cell lines would yield an inventory of the building blocks of the most commonly used systems in biological research. Mass spectrometry-based proteomics can identify and quantify proteins in a global and unbiased manner and can highlight the cellular processes that are altered between such systems. We analyzed 11 human cell lines using an LTQ-Orbitrap family mass spectrometer with a "high field" Orbitrap mass analyzer with improved resolution and sequencing speed.

Disclosure of erlotinib as a multikinase inhibitor in pancreatic ductal adenocarcinoma.

A placebo-controlled phase 3 trial demonstrated that the epidermal growth factor receptor (EGFR) inhibitor erlotinib in combination with gemcitabine was especially efficient in a pancreatic ductal adenocarcinoma (PDAC) subgroup of patients developing skin toxicity. However, EGFR expression was not predictive for response, and markers to characterize an erlotinib-responding PDAC group are currently missing. In this work, we observed high erlotinib IC50 values in a panel of human and murine PDAC cell lines.

Analysis of phosphoproteomics data.

Regulation of protein phosphorylation plays an important role in many cellular processes, particularly in signal transduction. Diseases such as cancer and inflammation are often linked to aberrant signaling pathways. Mass spectrometry-based methods allow monitoring the phosphorylation status in an unbiased and quantitative manner.

Identifying differentially regulated subnetworks from phosphoproteomic data.

Background: Various high throughput methods are available for detecting regulations at the level of transcription, translation or posttranslation (e.g. phosphorylation).

Probing teichoic acid genetics with bioactive molecules reveals new interactions among diverse processes in bacterial cell wall biogenesis.

The bacterial cell wall has been a celebrated target for antibiotics and holds real promise for the discovery of new antibacterial chemical matter. In addition to peptidoglycan, the walls of Gram-positive bacteria contain large amounts of the polymer teichoic acid, covalently attached to peptidoglycan. Recently, wall teichoic acid was shown to be essential to the proper morphology of Bacillus subtilis and an important virulence factor for Staphylococcus aureus.

Prediction of mechanisms of action of antibacterial compounds by gene expression profiling.

We have generated a database of expression profiles carrying the transcriptional responses of the model organism Bacillus subtilis following treatment with 37 well-characterized antibacterial compounds of different classes. The database was used to build a predictor for the assignment of the mechanisms of action (MoAs) of antibacterial compounds by the use of support vector machines. This predictor was able to correctly classify the MoA class for most compounds tested.