December Meeting

Speaker: Dingyin Tao, National Center for Advancing Translational Sciences, NIH

Topic: From Mass Spectrometry-Based Proteomics to Patient Care

Date: December 11, 2023

Time: 6:15 pm Dinner, 7:15 pm Presentation

Location: Shimadzu Scientific Instrument, Inc. Training Center 7100 Riverwood Drive, Columbia, MD 21046 (Directions)

Dinner: Please RSVP to Jonathan Ferguson (jonathan.ferguson33@gmail.com) by Friday, December 8 if you will be attending the dinner.

Abstract: Mass spectrometry-based proteomics plays a vital role in various stages of translational science, e.g. biomarker discovery and drug target identification. I will discuss these two separate cases: the first part focuses on the discovery of 35 parasite markers from which we selected a single candidate for use in a point-of-need (PON) rapid diagnostic test. We performed a cross-sectional, multi-omics study of saliva from 364 children with subclinical infection in Cameroon and Zambia, and produced a prototype saliva-based PON lateral flow immunoassay test for P. falciparum gametocyte carriers. This assay is being commercialized by a company founded in South Africa. The second part focuses on a new drug target for an old drug, Auranofin. UBA1 is the primary E1 ubiquitin-activating enzyme responsible for generation of activated ubiquitin required for ubiquitination, a process that regulates stability and function of numerous proteins. Abnormal UBA1 activity or ubiquitination causes or drives many human diseases, such as cancer, major neurodegenerative diseases, Angelman syndrome, VEXAS syndrome, and spinal muscular atrophy, as well as aging, highlighting the importance of the discovery of small molecule modulators of UBA1 activity for research and therapeutic purposes. Auranofin, a drug approved for the treatment of rheumatoid arthritis, was found in our high-throughput compound screen assay. Furthermore, HPLC-MS/MS was used to identify UBA1 as the new drug target of Auranofin. Through proteomics analysis, it was confirmed that auranofin binds to UBA1’s ubiquitin fold domain and conjugates to the Cys 1039 residue. This binding enhances interactions of UBA1 with at least 20 different E2 ubiquitin-conjugating enzymes, facilitating ubiquitin charging to E2 and increasing the activities of seven representative E3s in vitro. Auranofin promotes ubiquitination and degradation of misfolded ER proteins during ER-associated degradation in cells at low nanomolar concentrations. It also facilitates outer mitochondrial membrane-associated degradation. These findings suggest auranofin can serve as a much-needed tool for UBA1 research and therapeutic exploration.
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WBMSDG is including a lightning talk before the main talk at each event. Each lightning talk lasts 7 minutes with no more than 5 slides, and 5 minutes for Q&A. The desired speakers are early-career researchers/scientists in mass spec-related areas. They can be junior scientists, postdoctoral fellows, graduate or undergraduate students.

Please submit abstracts to co-chairs to apply for the lightning talks, and our board members will review the abstracts on a rolling basis.

Structure the abstract (maximum 300 words) with the following headings:
Title (maximum 20 words)
Authors and affiliations (Including senior authors/PIs)
Introduction
Methods
Results
Conclusions

November Meeting

Speaker: Kristine Glunde, Johns Hopkins University

Topic: Toward MALDI Microscopy: FluoMALDI, RaMALDI, and Single Cells

Date: November 13, 2023

Time: 6:15 pm Dinner, 7:15 pm Presentation

Location: Shimadzu Scientific Instrument, Inc. Training Center 7100 Riverwood Drive, Columbia, MD 21046 (Directions) This will be an in-person meeting.

Dinner: Please RSVP to Jonathan Ferguson (jonathan.ferguson33@gmail.com) by Friday, November 10 if you will be attending the dinner.

Abstract: The spatial resolution of matrix-assisted laser desorption/ionization (MALDI) imaging has significantly improved over the years and has now reached single cell resolution on commercial imaging mass spectrometers. This opens new possibilities of combining MALDI imaging with optical microscopy technologies including Raman and fluorescence microscopy. This talk will discuss our recently developed RaMALDI and FluoMALDI applications, which are streamlined, integrated, multimodal imaging workflows of Raman and fluorescence microscopy with MALDI imaging, performed on a single tissue section with one sample preparation protocol. In these projects, we discovered that co-crystallization of fluorophores with MALDI matrices significantly enhances fluorophore brightness, enabling the amplification of innate tissue autofluorescence and exogenous fluorophores. FluoMALDI and RaMALDI will advance structural-functional microscopic imaging in cell biology, biomedicine, and pathology.
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WBMSDG has decided to include a lightning talk before the main talk at each event. Each lightning talk lasts 7 minutes with no more than 5 slides, and 5 minutes for Q&A. The desired speakers are early-career researchers/scientists in mass spec-related areas. They can be junior scientists, postdoctoral fellows, graduate or undergraduate students.

Please submit abstracts to co-chairs to apply for the lightning talks, and our board members will review the abstracts on a rolling basis.

Structure the abstract (maximum 300 words) with the following headings:
Title (maximum 20 words)
Authors and affiliations (Including senior authors/PIs)
Introduction
Methods
Results
Conclusions

October Meeting

Speaker: Bill McDonough, University of Maryland

Topic: I didn’t know you could do that

Date: Monday, October 16, 2023

Time: 6:15 pm Dinner, 7:15 pm Presentation

Location: Shimadzu Scientific Instrument, Inc. Training Center 7100 Riverwood Drive, Columbia, MD 21046 (Directions) This will be an in-person meeting.

Dinner: Please RSVP to Jonathan Ferguson (jonathan.ferguson33@gmail.com) by Friday, October 13 if you will be attending the dinner.

Abstract: When I started out in the mass spectrometry business, I didn’t have any background (undergraduate degree in anthropology). However, by the end of my PhD, I was pretty good at analyzing samples on a thermal ionization mass spectrometer (TIMS). However, ignorance was an advantage, and nobody told me what I could and could not do. We bought bits and pieces, put them together, and we learned how to do things; we called our friends, and I asked lots of questions. The best two things that happened (1) we had good friends, and (2) we got a lot of money to start out with. We were not entirely stupid, but we were naïve. That helps a lot. Tonight, I’ll talk about a 30 year journey of fun LA-ICPMS accomplishments by our team that covers nuclear forensic, cutting-edge geochemistry, whodunit poisoning stories, pollution in the region, and getting ready for working on the Moon.
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Our WBMSDG board has decided to include two lightning talks before the main talk starting on our October event. Each lightning talk lasts 7 minutes with no more than 5 slides, and 5 minutes for Q&A for both talks. The desired speakers are early-career researchers/scientists in mass spec-related areas. They can be junior scientists, postdoctoral fellows, graduate or undergraduate students.

Please submit abstracts to co-chairs to apply for the lightning talks, and our board members will review the abstracts on a rolling basis.

Structure the abstract (maximum 300 words) with the following headings:
Title (maximum 20 words)
Authors and affiliations (Including senior authors/PIs)
Introduction
Methods
Results
Conclusions

September 2023 Meeting and Vendor Night

Speaker: John R. Yates, The Scripps Research Institute

Topic: How a single mutation in CFTR causes the systemic disease cystic fibrosis: interactions, PTMs, and structure

Date: Monday, September 11, 2023

Time: 6:00 pm Dinner and Vendor Night, 7:15 pm Presentation

Location: Shimadzu Scientific Instrument, Inc. Training Center 7100 Riverwood Drive, Columbia, MD 21046 (Directions) This will be an in-person meeting.

Dinner: Please RSVP to Jonathan Ferguson (jonathan.ferguson33@gmail.com) by Friday, September 8 if you will be attending the dinner.

Abstract: Protein conformation is dynamic as it is influenced by post-translational modifications (PTMs) and interactions with other proteins, small molecules or RNA, for example. However, in vivo characterization of protein structures and protein structural changes after perturbation is a major challenge. Therefore, experiments to characterize protein structures are typically performed in vitro and with highly purified proteins or protein
complexes, revealing a static picture of the protein. To identify the true conformational space occupied by proteins in vivo, we developed a novel low-resolution method named Covalent Protein Painting (CPP) that allows the characterization of protein conformations in vivo. Here, we report how an ion channel, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), is conformationally changed during biogenesis and channel opening in the cell. Our study led to the identification of a novel opening mechanism for CFTR by revealing that the interaction of the intracellular loop 2 (ICL2) with the nucleotide binding domain 2 (NDB2) of CFTR is needed for channel gating, and this interaction occurs concomitantly with changes to the narrow part of the pore and the walker A lysine in NBD1 for wt CFTR. However, the ICL2:NBD2 interface, which forms a “ball-in-a-socket” motif, is uncoupled during biogenesis, likely to prevent inadvertent channel activation during transport. Mutation of K273 in the ICL2 loop severely impaired CFTR biogenesis and led to accumulation of CFTR in the Golgi and TGN. CPP further revealed that, even upon treatment with current approved drugs such as Trikafta or at permissive temperature, the uncoupled state of ICL2 is a prominent feature of the misfolded CFTR mutants ∆F508 and N1303K that cause Cystic Fibrosis (CF). Although Trikafta treatment reduced the amount of uncoupled ICL2:NBD2 interfaces, more than 75% of F508 CFTR remained in the uncoupled state, suggesting that stabilization of this interface could produce a more efficient CF drug. CPP can characterize a protein in its native environment and measure the effect of complex PTMs and protein interactions on protein structure, making it broadly applicable and valuable for the development of new therapies.
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The Washington-Baltimore Mass Spectrometry Discussion Group is pleased to announce that our first event of 2023-2024 will be kicked off on September 11, 2023 (Monday) at Shimadzu Scientific in Columbia, MD. It is our true privilege this year that a giant in our field, Dr. John Yates III from Scripps Research, will be speaking as the keynote speaker at this event.

Our WBMSDG board has decided to include two lightning talks before the main talk starting with our October event. Each lightning talk lasts 7 minutes with no more than 5 slides, and 5 minutes for Q&A for both talks. The desired speakers are early-career researchers/scientists in mass spec-related areas. They can be junior scientists, postdoctoral fellows, graduate or undergraduate students.

Please submit abstracts to co-chairs to apply for the lightning talks, and our board members will review the abstracts on a rolling basis.

Structure the abstract (maximum 300 words) with the following headings:
Title (maximum 20 words)
Authors and affiliations (Including senior authors/PIs)
Introduction
Methods
Results
Conclusions

(We are updating our sponsors list for 2023-2024.)

May Meeting

Speaker: Aleksandra Nita-Lazar, NIAID, NIH

Topic: Macrophage signaling mechanisms: deciphering protein networks

Date: Monday, May 15, 2023

Time: 6:15 pm Dinner, 7:15 pm Presentation

Location: Shimadzu Scientific Instrument, Inc. Training Center 7100 Riverwood Drive, Columbia, MD 21046 (Directions)
This will be an in-person meeting. Attendees are required to show a vaccine card (either at the door or in advance using the web form) . If you have submitted your vaccine card before, your status is already recorded.

Dinner: Please RSVP to Andy Qi (andy.yue.qi@gmail.com) by Friday, May 12 if you will be attending the dinner.

Abstract: Toll-like receptor (TLR) signaling in macrophages is essential for generating effective innate immune responses. Quantitative differences dependent on the dose and timing of the stimulus critically affect cell function and often involve proteins that are not components of widely shared transduction pathways. Mathematical modeling is an important approach to better understand how these signaling networks function in time and space. We have successfully modeled the S1P signaling pathway in macrophages using selected reaction monitoring (SRM) to measure the absolute abundance of the pathway proteins. The resulting values became parameters in a computational pathway model. To model the TLR signaling networks, we developed assays for the canonical
TLR signaling pathway and related proteins and phosphoproteins and used parallel reaction monitoring (PRM) with heavy-labeled internal peptide standards to quantify protein and phosphorylated protein molecule numbers per cell in untreated and LPS-stimulated macrophages. The absolute protein abundance values were entered into a model of the TLR pathway developed using Simmune, the rule-based modeling tool with a visual interface. To reach beyond basal level quantification, the TLR signaling network model is tested further and combined with global proteomic approaches to discover
biologically important new proteins, protein complexes and PTMs involved in this innate immune pathway. The protein and PTM levels are quantified in macrophages under diverse, but well-defined conditions (different TLR ligands, whole pathogens, and cells with mutations in specific signaling molecules). These data will allow to parameterize and test the TLR network model under a variety of conditions. Together, the interconnected projects will lead to the better understanding how the immune signaling pathways are regulated and activated during an infection. This research was supported by the Intramural Research Program of NIAID, NIH.

Thank you to our 2022-2023 sponsors!