Session MOC. There are 4 abstracts in this session.



Session: CELL BIOLOGY: PROTEOMIC ANALYSIS OF PTMS AND PROTEOFORMS, time: 09:50 AM - 10:15 AM

Novel Top-Down Proteomics Strategies for Comprehensive Analysis of Proteoforms

Ying Ge

Top-down mass spectrometry (MS)-based proteomics is arguably the most powerful method to comprehensively characterize proteoforms that arise from genetic variations, alternative splicing, and post-translational modifications (PTMs), but myriad challenges remain. We have been developing novel strategies to address the challenges in top-down proteomics in a multi-pronged approach. To address the protein solubility challenge, we have recently identified a photo-cleavable anionic surfactant (“Azo”) that can be rapidly degraded upon UV irradiation, for top-down proteomics. Azo is MS-compatible and can effectively solubilize proteins with performance comparable to SDS. Importantly, Azo-aided top-down proteomics enables the solubilization of membrane proteins for comprehensive characterization of PTMs. To address the proteome complexity challenge, we have been developing novel strategies for multi-dimensional liquid chromatography (MDLC) to separate intact proteins. We developed novel hydrophobic interaction chromatography (HIC) materials for high-resolution separation of intact proteins under non-denaturing mode and demonstrated the potential of online HIC/MS for top-down proteomics. Given the difficulty in detecting large proteins in top-down MS, importantly, we developed a serial size exclusion chromatography strategy for size-based protein separation that can be coupled with online reverse phase chromatography and high-resolution MS which enabled the top–down MS analysis of large proteins (>200 kDa). Furthermore, we established a robust top-down LC/MS-based targeted proteomics platform for quantification of protein expression and PTMs concurrently in complex mixtures with high throughput and high reproducibility. To address the proteome dynamic range, we have been developing novel nanomaterials that can bind low abundance proteins and PTMs with high specificity. We developed an intergrated top-down phosphoproteomics work flow that coupled phosphoprotein enrichment by functionalized nanoparticles with online top-down LC/MS/MS to enrich, identify, quantify, and charaterize intact phosphoproteins directly from cell lysates and tissue homogenates. We have also recently successfully enriched low abundance proteins from blood for comprehensive analysis of proteoforms by top-down proteomics.
Tips and Tricks:

Session: CELL BIOLOGY: PROTEOMIC ANALYSIS OF PTMS AND PROTEOFORMS, time: 10:15 AM - 10:40 AM

Profiling system dynamics and crosstalk between the ubiquitin and Nedd8 systems

Donald Kirkpatrick

Ubiquitin (Ub) and ubiquitin-like (UBL) proteins play a critical role in regulating dynamic cellular processes. Most notably, the Ub system functions to sculpt the cellular proteome through proteasomal degradation and autophagy. In addition, post-translational modification of proteins by Ub and UBLs can alter their activity, localization and interaction partners. A family of enzymes operating at this critical intersection are the Cullin-RING E3 ligase (CRL) complexes that promote ubiquitination and degradation of numerous substrates. Activation of a CRL complex requires post-translational modification of the cullin subunit by the UBL protein Nedd8, which in turn stabilizes association of the substrate recruitment module to permit efficient substrate recruitment. Despite recent advances, key unanswered questions remain. For example, what factors trigger incorporation of different substrate recruitment modules into functional CRL complexes? Also, do functionally relevant Nedd8 substrates exist besides the cullins? Recently developed mass spectrometry proteomics technologies shed light on these and other important questions. This talk will focus on two technologies that together elucidate the interplay between ubiquitination, neddylation, and the machineries controlling these interrelated processes. Protein Interaction Kinetics and Estimation of Stoichiometries (PIKES) analysis is a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Serial NEDD8-Ubiquitin Substrate Profiling (sNUSP) employs Nedd8-R74K knock-in cells and K-εGG-peptide enrichment following non-classical proteolysis to discriminate between endogenous NEDD8- and Ub-modification sites. Using these two approaches, we show that Cand1 is both a novel neddylation substrate as well as critical exchange factor required for efficient substrate degradation by CRL4 E3 ligases.
Tips and Tricks:

Session: CELL BIOLOGY: PROTEOMIC ANALYSIS OF PTMS AND PROTEOFORMS, time: 10:40 AM - 10:55 AM

Site-specific analysis of the Asp- and Glu-ADP-ribosylated proteome by quantitative mass spectrometry

Yonghao Yu
UT Southwestern Medical Center, Dallas, TX

ADP-ribosylation is a protein post-translational modification that is critically involved in a wide array of biological processes connected to cell stress responses. Enzymes known as poly-ADP-ribose polymerases (PARPs) catalyze the addition of the ADP-ribose units to amino acids with various side chain chemistries. In particular, the PARP family member PARP1 is responsible for the modification of a large number of proteins and is involved in initiation of the DNA damage response. Despite the progresses of the PARP1 inhibitors in the clinic (4 PARP1 inhibitors have been approved for the treatment of human cancer), the mechanisms through which PARP1 functions are still incompletely understood. The analysis of protein ADP-ribosylation is challenging because PARylation is a low-abundance, labile and heterogeneous protein modification. Recently, we developed an integrative proteomic platform for the site-specific analysis of protein ADP-ribosylation on Asp and Glu residues. Herein, we describe the method, and demonstrate its utility in quantitative characterization of the human Asp- and Glu-ADP-ribosylated proteome.

Tips and Tricks:

Session: CELL BIOLOGY: PROTEOMIC ANALYSIS OF PTMS AND PROTEOFORMS, time: 10:55 AM - 11:10 AM

EXoO-Tn: Tag-n-Map the Tn Antigen in the Human Proteome

Weiming Yang; Minghui Ao; Angellina Song; Yuanwei Xu; Lori Sokoll; Hui Zhang
Johns Hopkins University, Baltimore, MD

Tn antigen (Tn), a single N-acetylgalactosamine (GalNAc) monosaccharide attached to protein Ser/Thr residues, is found on most solid tumors yet rarely detected in adult tissues, featuring it one of the most distinctive signatures of cancer. Although it is important in cancer, Tn-glycosylation sites are not entirely clear owing to the lack of suitable technology. Knowing the Tn-glycosylation sites may empower the future development of prevention, diagnostics, and therapeutics of cancer and other diseases associated with the expression of Tn. Here, we introduce a technology named EXoO-Tn for large-scale mapping of Tn-glycosylation sites. EXoO-Tn utilizes glycosyltransferase C1GalT1 and isotopically-labeled UDP-Gal(13C6) to tag and convert Tn to Gal(13C6)-Tn, which gives rise to a unique glycan mass. This exquisite Gal(13C6)-Tn structure is then recognized by a human-gut-bacterial enzyme, called OpeRATOR, that cleaves N-termini of the Gal(13C6)-Tn-occupied Ser/Thr residues to yield site-containing glycopeptides. The two enzymes C1GalT1 and OpeRATOR could be used concurrently in a one-pot reaction. The effectiveness of EXoO-Tn was benchmarked by analyzing Jurkat cells, where 947 Tn-glycosylation sites from 480 glycoproteins were mapped. Bioinformatic analysis of the identified site-specific Tn-glycoproteins revealed conserved motif, cellular localization, the relative location of Tn in proteins. Given the importance of Tn in diseases, EXoO-Tn is anticipated to have broad utilities in the translational and clinical studies.

Tips and Tricks: