Session TOF. There are 4 abstracts in this session.

Session: Protein Proteoforms in Health and Disease, time: 4:30 - 4:55 pm

A PTM Code for Membrane Protein Maturation

John R. Yates1; Sandra Pankow1; Casimir Bamberger1; Diego Calzolari2; Salvador Martínez-Bartolomé1; Mathieu Lavallée-Adam3
1The Scripps Research Institute, La Jolla, CA; 2Qualcomm, San Diego, CA; 3University of Ottawa, Ottawa, Canada

A component to understanding biological processes involves identifying the proteins expressed in cells as well as their modifications and the dynamics of processes. This process has benefited from the sequencing of genomes, although this information Is not uncovered from DNA sequencing. Mass spectrometry together with informatic tools can uncover the type of modification and it’s a location in a peptide sequence.  Advances in multi-dimensional separations as well as mass spectrometry have improved the scale of experiments for protein identification. This has improved the analysis of protein complexes, and more complicated protein mixtures. Quantitative mass spectrometry has also helped to determine the role of modifications in regulating biological processes. Using the loss of function mutant form of the Cystic Fibrosis Transport Regulator (DF508) as it progresses through the folding pathway as a model to understand the regulation of protein maturation, we have discovered a post translational modification code that regulates the maturation of CFTR. This has provided a better understanding of the loss of function associated with mutation.1,2We are now exploring if this code is general for membrane protein maturation.  

1Pankow et al Nature2015, 528, 510-6., 2Pankow et al Science Signaling 2019, 12(562) eaan7984.

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Session: Protein Proteoforms in Health and Disease, time: 4:55 - 5:20 pm

Polycomb loss mediated reprogamming of the epigenome drives oncogenesis in malignant peripheral nerve sheath tumors

John B. Wojcik; Dylan M. Marchione; Simone Sidoli; Benjamin A. Garcia
University of Pennsylvania School of Medicine, Philadelphia, PA

Malignant peripheral nerve sheath tumor (MPNST) is an aggressive sarcoma with recurrent loss of function alterations in polycomb-repressive complex 2 (PRC2), a histone-modifying complex involved in transcriptional silencing. To understand the role of PRC2 loss in pathogenesis and identify therapeutic targets, we conducted parallel global epigenomic and proteomic analysis of archival formalin-fixed, paraffin-embedded human MPNSTs with and without PRC2 loss (MPNSTLOSS vs. MPNSTRET).  In MPNSTLOSS, histone post-translational modifications (PTMs) associated with active transcription, most notably H3K27Ac and H3K36me2, were increased and polycomb-mediated repressive H3K27 di- and trimethylation (H3K27me2/3) were globally lost, without a compensatory gain in other repressive PTMs.  Instead, DNA methylation was globally increased. Epigenomic changes were associated with upregulation of proteins in growth pathways and reduction in interferon signaling and antigen presentation, suggesting a role for epigenomic changes tumor progression and immune evasion, respectively. The epigenomic changes also resulted in therapeutic vulnerabilities. Knockdown of NSD2, the methyltransferase responsible for H3K36me2, restored MHC expression and induced interferon pathway expression in a similar manner to polycomb restoration. MPNSTLOSS were also highly sensitive to DNA methyltransferase inhibitors and HDAC inhibitors, suggesting that global loss of polycomb-mediated repression renders MPNSTLOSS differentially dependent on DNA methylation to maintain transcriptional integrity and thus highly sensitive to therapeutics that promote aberrant transcription initiation.

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Session: Protein Proteoforms in Health and Disease, time: 5:20 - 5:35 pm

Deciphering the Human Heart Proteoform Landscape in Cardiac Disease and Regeneration

Trisha Tucholski1; Ling Gao2; Zachery Gregorich1; Wenxuan Cai1; Kyle Brown1; Yanlong Zhu1; Bifan Chen1; Samantha Knott1; Andrew Alpert3; Jianyi Zhang2; Ying Ge1
1University of Wisconsin - Madison, Madison, Wisconsin ; 2University of Alabama at Birmingham, Birmingham, Alabama; 3PolyLC Inc., Columbia , Maryland

Heart diseases remain the leading cause of death in developed countries for both men and women. Altered post-translational modifications (PTMs) and variations in amino acid sequence for cardiac sarcomere proteins have been implicated as causative factors for cardiovascular diseases. Nevertheless, the disease mechanisms are highly heterogeneous and poorly understood. To begin to understand the molecular biology underlying human heart disease and cardiac regeneration, we must obtain a global qualitative and quantitative view of proteoform landscape with critical knowledge on the combinatorial PTM-amino acid sequence variants. Mass spectrometry (MS)-based top-down proteomics (TDP) is the most powerful technology for deciphering PTM codes together with amino acid sequence variations, providing essential insight into the structure and function of proteoforms, the effectors of all biological processes. Herein, we seek to develop and implement novel TDP tools to qualitatively and quantitatively characterize human heart proteoforms to deepen our understanding of heart health and disease. Using a proteomics platform, which combines serial size-exclusion chromatography with high-resolution top-down MS, we have gained access to the high-molecular weight portion of the human heart proteome and identified previously unknown phosphoproteins in the cardiac sarcomere. Using quantitative top-down proteomics, we have unveiled a reversal of deleterious PTM changes following cardiac injury and treatment with an induced pluripotent stem cell (iPSC) – derived patch. Additionally, a novel quantitative proteomics platform developed in our lab has allowed us to use top-down LC-MS to assess expression-level changes of proteins in the cardiac sarcomere following iPSC-patch therapy. Combined with global label-free proteomics, we uncover the proteome-level changes in a swine model for iPSC-patch therapy and cardiac regeneration.

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Session: Protein Proteoforms in Health and Disease, time: 5:35 - 5:50 pm

Cell Type-Resolved Analysis of Blood Proteoforms by Large-Scale Top-Down Proteomics

Paul Thomas1; R. Vince Gerbasi1; Rafael Melani1; Jacek Sikora1; Timothy Toby1; Kristina Srzentic1; Luca Fornelli1, 2; Richard LeDuc1; Josiah Hutton1, 3; Ryan Fellers1; Joseph Greer1; Jeannie Camarillo1; Lissa Anderson4; Chris Hendrickson4; Neil Kelleher1
1Northwestern University, Evanston, IL; 2University of Oklahoma, Norman, OK; 3Princeton University, Princeton, NJ; 4NHMFL, Tallahassee, FL

Proteoforms have emerged as a new unit of measurement in proteomics that is strongly connected to protein function and organismal phenotypes.  While many protein catalogs have been created over the years, few contain and report on the proteoforms present.  Here, we will explore the rationale for cataloging proteoforms in health and disease and describe challenges and solutions that arise when performing top-down proteomics “at-scale.” 

Blood is a suspension of cells in plasma that circulates throughout the human body.  It is responsible for transport of proteins and metabolites as well as mounting immune responses to external threats.  Techniques such as fluorescence activated cell sorting (FACS) and immunocapture can be used to isolate specific subsets of cells for further analysis.  Here, we used FACS and positive and negative immunopurification to isolate cell populations. Next, we extract and analyze intact proteoforms from these multiple, diverse cell types from both the lymphoid and myeloid lineage to examine their proteoform content and establish cell-type signatures.

Top-Down proteomics is a technique that allows users to catalog full, intact proteoforms by omitting the protease digestion used in standard proteomics workflow.  To this point, most top-down proteomics experiments have been limited in scope, largely due to the many challenges encountered in the acquisition and analysis of very large top-down datasets (>100s of files).  This project required the development of novel solutions to these challenges including robust sample preparation workflows, cloud-based solutions for high-throughput analysis of top-down proteomics data, and algorithms for accurate false-discovery rate estimation.  With these challenges met, robust, reproducible sample-to-answer workflows can be established for top-down proteomics.  At full scale, an atlas of proteoforms from healthy blood will help researchers in the future to understand health and disease at proteoform-level resolution.

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