Faculty Bibliography

Although genetically compact, HIV-1 commandeers vast arrays of cellular machinery to sustain and protect it during cycles of viral outgrowth. Transposon-mediated saturation linker scanning mutagenesis was used to isolate fully replication-competent viruses harbouring a potent foreign epitope tag. Using these viral isolates, we performed differential isotopic labelling and affinity-capture mass spectrometric analyses on samples obtained from cultures of human lymphocytes to classify the vicinal interactomes of the viral Env and Vif proteins as they occur during natural infection. Importantly, interacting proteins were recovered without bias, regardless of their potential for positive, negative or neutral impact on viral replication. We identified specific host associations made with trimerized Env during its biosynthesis, at virological synapses, with innate immune effectors (such as HLA-E) and with certain cellular signalling pathways (for example, Notch1). We also defined Vif associations with host proteins involved in the control of nuclear transcription and nucleoside biosynthesis as well as those interacting stably or transiently with the cytoplasmic protein degradation apparatus. Our approach is broadly applicable to elucidating pathogen-host interactomes, providing high-certainty identification of interactors by their direct access during cycling infection. Understanding the pathophysiological consequences of these associations is likely to provide strategic targets for antiviral intervention.

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.

INTRODUCTION: Optic gliomas are classified as pilocytic astrocytoma (PA) or pilomyxoid astrocytoma (PMXA). Abundant bluish chondroid myxoid matrix is characteristic of PMXA but not PA. We sought to investigate the molecular composition of myxoid matrix and its biologic role in angiogenesis of optic gliomas. We reviewed clinical and pathological data on a cohort of 120 patients with optic glioma diagnosed at NYU Langone Medical Center from 1996 to 2014. We analyzed microvascular density (MVD), perfusion, hypoxia and proliferation by immunohistochemistry and ultrastructural features by electron microscopy. To identify the composition of the myxoid matrix in PMXA we performed liquid chromatography-mass spectrometry (LC-MS) without sample fractionation quantified using peptide spectral counts. PMXA showed significantly lower MVD by CD34 (8.1 vs 14.5, p-value < 0.002) and Erg (7 vs. 13.6, p-value 0.003) than PA, however GLUT-1 showed equal perfusion. Electron microscopy showed that PMXA contain both regular blood vessels with endothelial lining and channels completely lacking endothelial and smooth muscle cells. LC-MS stratified optic gliomas into three distinct groups. We identified 5389 proteins of which 188 were differentially expressed in the three groups (p<0.05, Benjamini-Hochberg adjustment). Between PA and PMXA, we found that most of differentially expressed proteins (146/188) displayed a positive fold change (increasing in PMXA relative to PA), and a minority (42/188) showed a negative fold change. The most abundant extracellular matrix proteins were a chondroitin sulfate proteoglycan versican (VCAN 3.7-fold increase Q=0.000463) and its paralog vertebrate Hyaluronan And Proteoglycan Link Protein 1 (HAPLN1, 22-fold increase from the PA to the PMXA group Q=4.60x10-7). Optic gliomas can develop endothelium-independent channels reminiscent of those in invertebrates to maintain blood supply. The myxoid matrix is composed of VCAN and its linking paralog HAPLN1. Targeting the myxoid matrix may provide novel avenues for therapy of optic gliom

LINE-1 (L1) retrotransposons are catalysts of evolution and disease whose sequences comprise a significant proportion of the human genome. L1 ribonucleoprotein particles incorporate a combination of permissive host factors that are essential to their lifecycle as well as repressive factors that constitute defenses against L1's mutagenic activity. We previously characterized host proteins associated with human L1 retrotransposons, as expressed in cell culture, using a combination of techniques including metabolic labeling and affinity proteomics. To build on these analyses, we have executed a suite of quantitative proteomic comparisons, yielding interactomic maps of affinity isolated L1s. These studies have revealed the presence of at least two populations of putative transposition intermediates that may exhibit distinctive intracellular localizations. We report the proteins partitioning within these distinct L1 populations and associated in vitro activities. Our observations provide a basis for the classification of L1 interactors into physical and functional modules and have enabled the development of in vitro systems to study L1 activity

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.

Large scale proteomics have made it possible to broadly screen samples for the presence of many types of post-translational modifications, such as phosphorylation, acetylation, and ubiquitination. This type of data has allowed the localization of these modifications to either a specific site on a proteolytically generated peptide or to within a small domain on the peptide. The resulting modification acceptor sites can then be mapped onto the appropriate protein sequences and the information archived. This paper describes the usage of a very large archive of experimental observations of human post-translational modifications to create a map of the most reproducible modification observations onto the complete set of human protein sequences. This set of modification acceptor sites was then directly translated into the genomic coordinates for the codons for the residues at those sites. We constructed the database g2pDB using this protein-to-codon site mapping information. The information in g2pDB has been made available through a RESTful-style API, allowing researchers to determine which specific protein modifications would be perturbed by a set of observed nucleotide variants determined by high throughput DNA or RNA sequencing.

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.

Antibodies made in large animals are integral to many biomedical research endeavors. Domesticated herd animals like goats, sheep, donkeys, horses and camelids all offer distinct advantages in antibody production. However, their cost of use is often prohibitive, especially where poor antigen response is commonplace; choosing a non-responsive animal can set a research program back or even prevent experiments from moving forward entirely. Over the course of production of antibodies from llamas, we found that some animals consistently produced a higher humoral antibody response than others, even to highly divergent antigens, as well as to their standard vaccines. Based on our initial data, we propose that these "high level responders" could be pre-selected by checking antibody titers against common vaccines given to domestic farm animals. Thus, time and money can be saved by reducing the chances of getting poor responding animals and minimizing the use of superfluous animals.

Selenocysteine is a naturally occurring proteogenic amino acid that is encoded in the genomic sequence of relatively abundant proteins in many of the model species commonly used for biomedical research. On the basis of an analysis of publicly available proteomics information, it was discovered that peptides containing selenocysteine were not being identified in tandem mass spectrometry proteomics data. Once the chemical basis for this exclusion was understood, a simple alteration in search parameters led to the confident identification of selenocysteine containing peptides from existing proteomics data, with no change in experimental protocols required.