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. We believe that the strategy delineated here provides a path forward to avoid the time- and cost-consuming step of having to develop immunological reagents against unproven targets. We expect that mass spectrometry-based platforms may become part of a rational process to rapidly test and qualify large number of candidate biomarkers to identify the few that stand a chance for further development and validation.

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. PDE10 inhibition restored corticostriatal input and boosted cortically driven indirect pathway activity. Cyclic nucleotide signaling is impaired in HD models, and PDE10 loss may represent a homeostatic adaptation to maintain signaling. Elevation of both cAMP and cGMP by PDE10 inhibition was required for rescue. Phosphoproteomic profiling of striatum in response to PDE10 inhibition highlighted plausible neural substrates responsible for the improvement. Early chronic PDE10 inhibition in Q175 mice showed improvements beyond those seen with acute administration after symptom onset, including partial reversal of striatal deregulated transcripts and the prevention of the emergence of HD neurophysiological deficits. VIDEO ABSTRACT.

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.

Use of colloidal silica-beads for the isolation of cell-surface proteins for mass spectrometry-based proteomics.

Chaney and Jacobson first introduced the colloidal silica-bead protocol for the coating of cellular plasma membranes in the early 1980s. Since then, this method has been successfully incorporated into a wide range of in vitro and in vivo applications for the isolation of cell-surface proteins. The principle is simple - cationic colloidal silica microbeads are introduced to a suspension or monolayer of cells in culture. Electrostatic interactions between the beads and the negatively charged plasma membrane, followed by cross-linking to the membrane with an anionic polymer, ensure attachment and maintain the native protein conformation. Cells are subsequently ruptured, and segregation of the resulting plasma membrane sheets from the remaining- cell constituents is achieved by ultracentrifugation through density gradients. The resulting membrane-bead pellet is treated with various detergents or chaotropic agents (i.e., urea) to elute bound proteins. If proteomic profiling by mass spectrometry is desired, proteins are denatured, carbamidomethylated, and digested into peptides prior to chromatography.

In-depth proteomics of ovarian cancer ascites: combining shotgun proteomics and selected reaction monitoring mass spectrometry.

Epithelial ovarian cancer (EOC) is the most common gynecological cancer and the ninth most common cancer overall. Major problems associated with EOC include poorly characterized disease progression, disease heterogeneity, lack of early detection markers and the development of chemoresistance. Early detection and treatment of EOC would significantly benefit from routine screening tests on available biofluids. We built on our experience in analyzing ovarian cancer ascites and present an analysis pipeline that combines discovery-based proteomics, bioinformatics prioritization and targeted proteomics quantification using Selected Reaction Monitoring Mass Spectrometry (SRM-MS). Ascitic fluids from patients with serous-type epithelial ovarian cancer were analyzed using comprehensive shotgun proteomics and compared to noncancerous ascitic fluids from patients with benign ovarian tumors. Integration of our data with published mRNA transcriptomic and proteomic data sets led to a panel of 51 candidate proteins. Systematic SRM-MS assay development was performed for a subset of these proteins using both synthetic peptides (13 proteins) and stable isotope labeled standards (4 proteins). Subsequently, precise relative quantification by stable isotope dilution-SRM (SID-SRM) in independent ascites and serum samples was performed as a proof-of-concept validation. The analysis strategy outlined here lays the foundation for future experiments using both larger numbers of patient samples and additional candidate proteins, and provides a template for the proteomics-based discovery of cancer biomarkers.

Targeted proteomics by selected reaction monitoring mass spectrometry: applications to systems biology and biomarker discovery.

Mass Spectrometry-based proteomics is now considered a relatively established strategy for protein analysis, ranging from global expression profiling to the identification of protein complexes and specific post-translational modifications. Recently, Selected Reaction Monitoring Mass Spectrometry (SRM-MS) has become increasingly popular in proteome research for the targeted quantification of proteins and post-translational modifications. Using triple quadrupole instrumentation (QqQ), specific analyte molecules are targeted in a data-directed mode. Used routinely for the quantitative analysis of small molecular compounds for at least three decades, the technology is now experiencing broadened application in the proteomics community. In the current review, we will provide a detailed summary of current developments in targeted proteomics, including some of the recent applications to biological research and biomarker discovery.

In-depth proteomic analyses of direct expressed prostatic secretions.

It is expected that clinically obtainable fluids that are proximal to organs contain a repertoire of secreted proteins and shed cells reflective of the physiological state of that tissue and thus represent potential sources for biomarker discovery, investigation of tissue-specific biology, and assay development. The prostate gland secretes many proteins into a prostatic fluid that combines with seminal vesicle fluids to promote sperm activation and function. Proximal fluids of the prostate that can be collected clinically are seminal plasma and expressed prostatic secretion (EPS) fluids. In the current study, MudPIT-based proteomics was applied to EPS obtained from nine men with prostate cancer and resulted in the confident identification of 916 unique proteins. Systematic bioinformatics analyses using publicly available microarray data of 21 human tissues (Human Gene Atlas), the Human Protein Atlas database, and other published proteomics data of shed/secreted proteins were performed to systematically analyze this comprehensive proteome. Therefore, we believe this data will be a valuable resource for the research community to study prostate biology and potentially assist in the identification of novel prostate cancer biomarkers. To further streamline this process, the entire data set was deposited to the Tranche repository for use by other researchers.

Isolation of cell surface proteins for mass spectrometry-based proteomics.

Defining the cell surface proteome has profound importance for understanding cell differentiation and cell-cell interactions, as well as numerous pathogenic abnormalities. Owing to their hydrophobic nature, plasma membrane proteins that reside on the cell surface pose analytical challenges and, despite efforts to overcome difficulties, remain under-represented in proteomic studies. Limitations in the classically employed ultracentrifugation-based approaches have led to the invention of more elaborate techniques for the purification of cell surface proteins. Three of these methods--cell surface coating with cationic colloidal silica beads, biotinylation and chemical capture of surface glycoproteins--allow for marked enrichment of this subcellular proteome, with each approach offering unique advantages and characteristics for different experiments. In this article, we introduce the principles of each purification method and discuss applications from the recent literature.

Peptide separations by on-line MudPIT compared to isoelectric focusing in an off-gel format: application to a membrane-enriched fraction from C2C12 mouse skeletal muscle cells.

High-resolution peptide separation is pivotal for successful shotgun proteomics. The need for capable techniques propels invention and improvement of ever more sophisticated approaches. Recently, Agilent Technologies has introduced the OFFGEL fractionator, which conducts peptide separation by isoelectric focusing in an off-gel setup. This platform has been shown to accomplish high resolution of peptides for diverse sample types, yielding valuable advantages over comparable separation techniques. In this study, we deliver the first comparison of the newly emerging OFFGEL approach to the well-established on-line MudPIT platform. Samples from a membrane-enriched fraction isolated from murine C2C12 cells were subjected to replicate analysis by OFFGEL (12 fractions, pH 3-10) followed by RP-LC-MS/MS or 12-step on-line MudPIT. OFFGEL analyses yielded 1398 proteins (identified by 10,269 peptides), while 1428 proteins (11,078 peptides) were detected with the MudPIT approach. Thus, our data shows that both platforms produce highly comparable results in terms of protein/peptide identifications and reproducibility for the sample type analyzed. We achieve more accurate peptide focusing after OFFGEL fractionation with 88% of all peptides binned to a single fraction, as compared to 61% of peptides detected in only one step in MudPIT analyses. Our study suggests that both platforms are equally capable of high quality peptide separation of a sample with medium complexity, rendering them comparably valuable for comprehensive proteomic analyses.