Session WOB. There are 4 abstracts in this session.

Session: Posttranslational Regulation, time: 09:50 - 10:15 am

The lysine acylation pathways as a bridge between metabolism and epigenetic mechanisms

Yingming Zhao
University Of Chicago, Chicago, IL

There are tens of thousands metabolites in mammalian cells. However, less than 1% of these metabolites’ non-metabolism functions have been characterized, representing a major knowledge gap in biology. In this presentation, we will report a new family of histone modifications that can be induced by short-chain lipid metabolites.  Using mass spectrometry and biochemical methods, we detected and comprehensively validated nine types of lysine short-chain, acylation pathways: propionylation, butyrylation, crotonylation, malonylation, succinylation, glutarylation, 2-hydroxyisobutyrylation, 3-hydroxybutyrylation, and benzoylation. These acylation pathways use short-chain CoAs as cofactors for their modification reactions that can be stimulated by their corresponding short-chain lipids.  We identified ~500 histone marks bearing the new PTMs. Using mass spectrometry-based proteomics approaches, we identified and quantified protein substrates bearing these modifications in core histones and non-histone proteins. We subsequently characterized the new PTM pathways by identifying their binding proteins and regulatory enzymes. Interestingly, some HDACs (e.g., SIRT5) have high activities to these new lysine acylations but not the widely studied lysine acetylation, suggesting that some of HDACs were mistakenly classified as deacetylases. These new PTM pathways are associated with gene expression, cellular physiology and diverse metabolic diseases. 

Tips and Tricks (if submitted):

Session: Posttranslational Regulation, time: 10:15 - 10:40 am

High-throughput Quantitative Top-down Proteomics for Deep Characterization of Intact Proteoforms with Post-Translational Modifications

Si Wu
University of Oklahoma, Norman, OK

The isobaric chemical tag labeling coupled with 2D pH RP/RPLC separation has been widely applied for the identification and quantification of peptides and proteins in bottom-up proteomics.  However, until recently, successfully applying these approaches to top-down proteomics has been limited.  Here we reported a high-throughput quantitative top-down platform with the following innovations: (1) developing a first proteome-level TMT top-down MS platform for quantifying intact proteoforms with the molecular weight (MW) of less than 35 kDa; (2) optimizing an offiline 2D pH RP/RPLC separation for top-down MS; (3) establishing an online 2D nano-UPLC system for small quantity sample analysis.  Applying this platform, we successfully identified 1400+ proteoforms in unlabeled E. coli cell lysate (5 μg total protein), and quantified 1000+ intact proteoforms in 15 μg TMT labeled proteins (i.e., E. coli and HeLa cell lysate). We observed that the detected signal to noise ratios were enhanced using the online 2DLC platform, which facilitate the identification of low abundance proteoforms with PTMs. To summarize, we have developed a highly effective technique for deep proteoform characterization and quantification in complex protein samples.

Tips and Tricks (if submitted):

Session: Posttranslational Regulation, time: 10:40 - 10:55 am

The LPS-responsive ADP-ribosylated proteome in primary human immune cells.

Casey Daniels; Arthur Nuccio; Aleksandra Nita-Lazar
NIAID, NIH, Bethesda,

Protein ADP-ribosylation is known to be an important regulatory element in DNA repair and cell death, cellular processes critical to cancer development. As such, PARP inhibitors have successfully been developed as chemotherapeutic agents and are now being used in the clinic to treat a growing range of cancers. These clinical applications have revealed that PARP inhibitors modulate the human immune system, and today PARP inhibitors are being combined with immunotherapy in human patients. This exciting new work is happening in the absence of a basic understanding of how ADP-ribosylation and PARP inhibition affects immune cells, a gap which we seek to address here using mass spectrometry-based proteomics. Our two databases reveal changes in the ADP-ribosylated proteome during activation of the innate immune response as modeled by the exposure of macrophages to lipopolysaccharide (LPS), a highly immunogenic molecule found on the outside of gram-negative bacteria. The first database analyzes human primary, mouse primary, and cell-line derived macrophages and describes LPS-responsive ADP-ribosylation events on over 1000 proteins. The second database gives a detailed view of the changes in primary human monocyte-derived macrophages, wherein over 3,000 sites from 1,400 proteins are robustly quantified during immune activation using isotopic labeling. Analysis of this data has revealed LPS-sensitive ADP-ribosylation of proteins known to regulate the MAP kinase cascade and NFκB signaling. We share our mechanistic studies related to these findings and discuss the implications of this work in our understanding of PAR, PARP and PARP inhibitor biology. This research was supported by the Intramural Research Program of NIAID, NIH.

Tips and Tricks (if submitted):

Session: Posttranslational Regulation, time: 10:55 - 11:10 am

Pathway-scale targeted mass spectrometry for high-resolution functional profiling of cell signaling

Paolo Cifani; Alex Kentsis
Sloan-Kettering Institute, New York, NY

In spite of extensive studies of cellular signaling, many fundamental processes such as pathway integration, cross-talk and feedback remain poorly understood. In addition, most observed oncogenic mutations are of unknown significance and precise and comprehensive measurements of cancer cell signaling remain elusive. To measure differential regulation of cellular biochemical activities, thus enabling precision medicine and cell biology, we have developed the Quantitative Cell Proteomics Atlas (QCPA, QCPA consists of panels of targeted mass spectrometry (MS) assays to determine abundance and stoichiometry of regulatory post-translational modifications of proteins covering most recurrently mutated and functionally relevant pathogenic pathways in human cells. QCPA currently profiles 1,944 peptides from 467 effectors of cell surface signaling, apoptosis, stress response, gene expression, quiescence, and proliferation. For each protein, QCPA includes peptides covering known post-translational regulatory sites to determine their stoichiometry, and independent unmodified peptides to measure protein abundance. These measurements are then used as surrogates of regulation and biochemical activities. We implemented QCPA using the recently developed accumulated ion monitoring (AIM), to achieve enrichment-free near-zeptomolar sensitivity of targeted MS detection for phosphorylated and unmodified peptides. This enabled precise and accurate quantitation of functionally modified proteins from clinically-accessible, microgram minute patient specimens, or as few as 10,000 cells. As a result, we were able to measure integrated multi-parametric signaling profiles, such as those induced by growth factor stimulation, serum starvation, and drug treatment. In particular, we determined oncogenic signaling induced by mutant FLT3 receptor tyrosine kinase in acute myeloid leukemia cells, and defined the mechanisms of susceptibility and resistance to new selective kinase inhibitors. The ability to precisely and accurately deploy thousands of targeted chemoproteomic assays per experiment enables a paradigm shift for comprehensive analysis of cell signaling. In addition, precision and robustness of this method are well suited for functional proteomics of clinical specimens.

Tips and Tricks (if submitted):