Faculty Bibliography

Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. Here we describe quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers, of which 77 provided high-quality data. Integrated analyses provided insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. Interrogation of the 5q trans-effects against the Library of Integrated Network-based Cellular Signatures, connected loss of CETN3 and SKP1 to elevated expression of epidermal growth factor receptor (EGFR), and SKP1 loss also to increased SRC tyrosine kinase. Global proteomic data confirmed a stromal-enriched group of proteins in addition to basal and luminal clusters, and pathway analysis of the phosphoproteome identified a G-protein-coupled receptor cluster that was not readily identified at the mRNA level. In addition to ERBB2, other amplicon-associated highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates the functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.

Every molecular player in the cast of biology's central dogma is being sequenced and quantified with increasing ease and coverage. To bring the resulting genomic, transcriptomic, and proteomic data sets into coherence, tools must be developed that do not constrain data acquisition and analytics in any way but rather provide simple links across previously acquired data sets with minimal preprocessing and hassle. Here we present such a tool: PGx, which supports proteogenomic integration of mass spectrometry proteomics data with next-generation sequencing by mapping identified peptides onto their putative genomic coordinates.

Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations and splice variants identified in cancer cells are translated. Herein we apply a proteogenomic data integration tool (QUILTS) to illustrate protein variant discovery using whole genome, whole transcriptome and global proteome datasets generated from a pair of luminal and basal-like breast cancer patient derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS sample process replicates defined here as an independent tandem MS experiment using identical sample material. Despite analysis of over thirty sample process replicates, only about 10% of SNVs (somatic and germline) detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNVs without a detectable mRNA transcript were also observed, suggesting that transcriptome coverage was incomplete (~80%). In contrast to germline variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than in the luminal tumor raising the possibility of differential translation or protein degradation effects. In conclusion, this large-scale proteogenomic integration allowed us to determine the degree to which mutations are translated and identified gaps in sequence coverage, thereby benchmarking current technology and progress towards whole cancer proteome and transcriptome analysis.

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal ( http://assays.cancer.gov ) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and posttranslational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

The field of proteogenomics has been driven by combined advances in next-generation sequencing (NGS) and proteomic methods. NGS technologies are now both rapid and affordable, making it feasible to include sequencing in the clinic and academic research setting. Alongside the improvements in sequencing technologies, methods in high throughput proteomics have increased the depth of coverage and the speed of analysis. The integration of these data types using continuously evolving bioinformatics methods allows for improvements in gene and protein annotation, and a more comprehensive understanding of biological systems.

Bullying is an intentional act that can wreak havoc in the life of an individual. With more than 1 billion users, YouTube is a powerful medium for disseminating information. The purpose of this study was to describe the extent to which content related to bullying is present on YouTube with respect to source, content, number of views, length, and year uploaded. Collectively, the videos in this sample were viewed more than half a billion times. The source of the most widely viewed videos was consumers, and none of the most widely viewed videos was posted by a governmental agency or a professional organization. The most common content in the videos was describing or depicting violence (n=89). Over one-half addressed getting help (n=56). Suicide was mentioned in 38 of the videos. Additional investment by professional agencies is warranted to improve understanding about ways to increase the dissemination of positive messages about bullying prevention, and about helping adolescents who are bullied on social media.

A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.-Gulati, S., Balderes, D., Kim, C., Guo, Z. A., Wilcox, L., Area-Gomez, E., Snider, J., Wolinski, H., Stagljar, I., Granato, J. T., Ruggles, K. V., DeGiorgis, J. A., Kohlwein, S. D., Schon, E. A., Sturley, S. L. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

OBJECTIVE: The recent Ebola virus disease (EVD) outbreak in 2014-2015 has been the largest and longest lasting to date. Media coverage about the outbreak has been extensive, but there are large gaps in our understanding of the ways in which widely accessed social media sites are used during times of public health crisis. The purpose of this study was to analyze widely viewed videos about EVD on the YouTube video-sharing site. METHODS: We coded the source, content, and characteristics of the 100 most widely viewed videos about EVD on YouTube. RESULTS: The videos included in the sample were viewed more than 73 million times. The death toll in West Africa was mentioned in nearly one-third of the videos. Over one-third of the videos mentioned how EVD was generally transmitted. There was little mention of treatment and no mention of the need for US funding of disaster preparedness; coordination between local, state, and federal governments; or beds ready for containment. No significant differences in the number of views were identified between video sources with the exception of a significantly higher number of views for "consumer videos" compared with "commercial television videos." CONCLUSIONS: With 1 billion unique users a month, YouTube has potential for both enhancing education and spreading misinformation. (Disaster Med Public Health Preparedness. 2015;0:1-5).

BACKGROUND: The purpose of this study was to describe the prevalence of aggressive and violent behaviors in the context of the school environment in a nationally representative sample of adolescent youth and to illustrate these patterns during 2001-2011. METHODS: We analyzed data from 84,734 participants via the Youth Risk Behavior Surveillance System (YRBSS). Frequencies were visualized using heatmaps. One-way analyses of variance and corresponding post hoc tests helped to identify if differences in prevalence fluctuated significantly across all years. RESULTS: Rates of youth feeling unsafe in their school environment, bringing weapons to school, and engaging in physical fighting on school property continue to persist. Findings illustrated that Hispanic youth and youth classified as "other" have emerged as particularly high-risk demographic subgroups over the past decade. Peer victimization and sexual victimization continue to affect girls disproportionately. CONCLUSIONS: Though some variation within demographic subgroups exists, rates of aggressive and violent behaviors in the context of the school environment continue to persist. Implications for the coordinated prevention of aggressive and violent behaviors among adolescent youth are discussed and recommendations for school-based prevention efforts are identified.

We describe integrated strategies that employ both translation of ENCODE data and major proteomic technology pillars to improve the identification of the missing proteins, protein isoforms, and PTMs. The results from proteoENCODEdb searches with experimental mass spectral data indicate that some novel splice forms detected at the transcript level are in fact translated to proteins. Our results provide a step toward the directives of the C-HPP initiative and related biomedical research.