Poster Session A

A.1 Dynamic proteomic profiling of the Salmonella-host interplay reveals new modes of action for known and novel virulence factors Jennifer Geddes-McAlister(1), Stefanie Vogt(2), Jennifer Rowland(2), Sarah Woodward(2), Arjun Sukumaran(1), Lilianne Gee(1), Baerbel Raupach(3), Brett Finlay(2), Felix Meissner(4) (1)University of Guelph, Guelph, Canada, (2)University of British Columbia, Vancouver, Canada, (3)Max Planck Institute for Infectious Biology, Berlin, Germany, (4)Max Planck Institute of Biochemistry, Martinsried, Germany Intracellular bacterial pathogens cause a diverse array of diseases in humans and represent a significant threat to global health. These pathogens have evolved sophisticated strategies including the secretion of virulence factors to interfere with host cell functions and to perturb immune responses. However, interplay between the host and pathogen at the protein level in the context of infection has not been systematically investigated. Our 'infectome' analysis aims to identify previously undescribed proteins involved in bacterial virulence and host immune defense, representing an opportunity to elucidate molecular mechanisms of host-pathogen interplay during disease. Here, we investigate the host-pathogen interplay between the pathogenic bacteria, Salmonella enterica serovar Typhimurium, and primary macrophages. We performed quantitative proteomics of the host cells infected with wild-type (SL1344) or the type 3 secretion system (T3SS) mutant strains (Dspi-1 and Dspi-2) in single runs using high resolution mass spectrometry on a Quadrupole Orbitrap instrument. Our results provide a comprehensive and dynamic view of both pathogen and host proteins during infection. In the host cells, we observed the upregulation of proinflammatory and lysosomal proteins, representing host defense mechanisms to initiate immune responses and combat bacterial invasion. For S. Typhimurium, integration of proteome and infectome data identified eight proteins not encoded on SPI-1 or SPI-2 as being co-regulated with known virulence factors, suggesting a co-functional role in virulence and infection. Additionally, murine model competitive index assays revealed virulence-associated phenotypes of five proteins and defined their roles in bacterial cell regulation, as well as their impact on the host proteome. Overall, we provide an innovative strategy for profiling infection from dual perspectives in a single assay and characterizing novel virulence factors. A.2 Identification of urine-derived diagnostic biomarkers for Tuberculosis Bridget Calder(1) (1)University of Cape Town, South Africa Tuberculosis remains a leading cause of death worldwide, driven in part by the lack of sufficiently decisive diagnostic tools in the clinical setting. In South Africa, the incidence of TB/HIV co-infection is high, and co-infected individuals have particularly bad clinical outcomes. Some of the available diagnostics have a sensitivity as low as 50% in HIV positive individuals, and sputum-based testing is not possible in a high proportion of TB positive patients. An alternative TB diagnostic test should have high sensitivity and specificity in TB/HIV co-infected individuals, and be applicable in a biofluid that is obtained non-invasively. Urine has been proposed as an ideal biofluid for these purposes, and previous studies have found biomarkers for renal or GIT diseases in human urine. Since disseminated or extrapulmonary TB is often found in HIV positive individuals post mortem, we theorised that it should be possible to find a signature for TB in human urine that is either of TB or human origin. To that end, we employed discovery mass spectrometry-based proteomics to survey the urine of individuals who had been classified into four clinical groups: TB+/HIV-, TB+/HIV+, TB-/HIV+, and TB-/HIV-. This is the largest human urinary proteome-based study to date, comprising 120 individuals. Using Random Forest machine learning, TB status could be predicted using only four human proteins with a sensitivity and specificity of 95% and 85%, respectively in a one third hold-out set of the total data. We propose these human-derived biomarkers as a potential diagnostic panel for TB, which warrants further validation in a larger cohort. A.3 Microscaled Proteogenomic Methods for Precision Oncology Shankha Satpathy(1), Eric Jaehnig(2), Karsten Krug(1), Michael Gillette(1), Alexander Saltzman(2), Kimberly Holloway(2), Meenakshi Anurag(2), Chen Huang(2), Purba Singh(2), Beom-Jun Kim(2), Goerge Miles(2), Noel Namai(2), Anna Malovannaya(2), DR Mani(1), Chuck Perou(3), Bing Zhang(2), Steven Carr(1), Matthew Ellis(2) (1)The Broad Institute of MIT and Harvard, (2)Baylor College of Medicine, (3)University of North Carolina Cancer proteogenomics combines genomics, transcriptomics and mass spectrometry-based proteomics to gain insights into cancer biology and treatment responsiveness. While proteogenomics analyses have shown great potential to deepen our understanding of cancer tissue complexity and signaling, how a patient's tumor changes upon treatment has largely been the province of genomics. This is due to technical difficulties associated with doing proteogenomic analysis on clinic-derived core-needle biopsies. To address this critical need, we have developed a “microscaled” proteogenomics approach for tumor-rich OCT-embedded core needle biopsies. Tissue-sparing specimen processing (“Biopsy Trifecta EXTraction”, BioTExt) and microscaled proteomics (MiProt) methodologies allowed generation of deep-scale proteogenomics datasets, with copy number and transcript information for >20,000 genes and mass spectrometry-based identification and quantification of nearly all expressed proteins in a tumor (>10,000 proteins) and more than >20,000 phosphosites starting with just 25 micrograms of peptides per sample. In order to understand the capabilities and limitations of our approach relative to conventional deepscale proteomics requiring >10X more starting material, we compared preclinical patient derived xenograft (PDX) models at conventional scale with data obtained by core-needle biopsy of the same tissues. Comparable depth and biological insights were obtained from the cores relative to surgically resected tumors. As a proof-of-concept for implementation in clinical trials, we applied microscaled proteogenomic methods to a small-scale clinical study where biopsies were accrued from patients with ERBB2+ advanced breast cancer before and 48 to 72 hours after the first dose of neoadjuvant Trastuzumab-based chemotherapy. Multi-omics comparisons were conducted between samples associated with residual disease versus samples associated with complete pathological response. Integrative proteogenomic analyses efficiently diagnosed the molecular bases of diverse candidate treatment resistance mechanisms including: 1) absence of ERBB2 amplification (false-ERBB2+); 2) insufficient ERBB2 activity for therapeutic sensitivity despite ERBB2 amplification (pseudo-ERBB2+); 3) resistance features in true-ERBB2+ cases including androgen receptor signaling, mucin expression and an inactive immune microenvironment; 4) lack of acute phospho-ERBB2 down-regulation in non-pCR cases. In summary, we have developed a proteogenomics pipeline well suited for large-scale cancer clinical studies to identify potential resistance mechanism in patients. We conclude that microscaled cancer proteogenomics could improve diagnostic precision in the clinical setting. A.4 Reduced proteasome activity in the aging brain results in ribosome stoichiometry loss and aggregation Joanna M. Kirkpatrick(1), Erika K. Sacramento(1), Mariateresa Mazzetto(1), Simone Di Sanzo(1), Cinzia Caterino(1), Aleksandar Bartolome(1), Michele Sanguanini(3), Nikoletta Papaevgeniou(4), Maria Lefaki(4), Dorothee Childs(5), Eva Terzibasi-Tozzini(2), Natalie Romanov(5), Mario Baumgart(1), Wolfgang Huber(5), Niki Chondrogianni(4), Michele Vendruscolo(3), Alessandro Cellerino(1,2), Alessandro Ori(1) (1)Leibniz Institute on Aging - Fritz Lipmann Institute (FLI), Jena, Germany, (2)Scuola Normale Superiore, Pisa, Italy, (3)Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Cambridge, UK, (4)Institute of Biology, Medicinal Chemistry and Biotechnology, Athens, Greece, (5)European Molecular Biology Laboratory, Heidelberg, Germany A progressive loss of protein homeostasis is characteristic of aging and a driver of neurodegeneration. To investigate this process quantitatively, we characterized proteome dynamics during brain aging by using the short-lived vertebrate Nothobranchius furzeri and combining transcriptomics, proteomics and thermal proteome profiling. We found that the correlation between protein and mRNA levels is progressively reduced during aging, and that post-transcriptional mechanisms are responsible for over 40% of these alterations. These changes induce a progressive stoichiometry loss in protein complexes, including ribosomes, which have low thermal stability in brain lysates and whose component proteins are enriched in aggregates found in old brains. Mechanistically, we show that reduced proteasome activity occurs early during brain aging, and is sufficient to induce loss of stoichiometry. Our work thus defines early events in the aging process that can be targeted to prevent loss of protein homeostasis and age-related neurodegeneration. A.5 Affinity Proteomics Reveals Assembly of PPP-type Phosphatase Holoenzyme by PPM1G-B56δ Parveen Kumar(1,2), Prajakta Tathe(1,2), Subbareddy Maddika(1) (1)Laboratory of Cell Death & Cell Survival, Centre for DNA Fingerprinting and Diagnostics, INDIA, (2)Graduate studies, Manipal Academy of Higher Education, Manipal 576104, INDIA Serine/threonine phosphatases form distinct holoenzymes to achieve substrate specificity. PPP serine/threonine phosphatase family members such as PP1 and PP2A are well known to assemble and function as holoenzymes, but none of the PP

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