Speaker: Bob Ernst, University of Maryland – Baltimore

Topic: Bacterial Lipids – Advances in Structural Analysis and Function using MALDI Mass Spectrometry

Date: March 17, 2025

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 Dingyin Tao (owendtao@gmail.com) by Friday, March 14 if you will be attending the dinner.

Abstract: Microbial membranes are specialized structures composed of complex lipids and proteins, with distinct lipid compositions characteristic of different microbes, such as bacteria, spirochetes, and fungi. The diversity of lipids makes generalizing their composition challenging, often resulting in unique profiles for particular genera, including Gram-positive and Gram-negative bacteria. These membrane lipids play crucial roles in cellular integrity, signaling, stress response, propagation, antibiotic resistance, speciation, and pathogenesis. Understanding the characteristics, structure, and mechanisms of bacterial lipid synthesis and regulation requiring cutting-edge technologies to enhance microbial lipidomics resolution. Advancements in instrumentation and extraction procedures have led to more sophisticated analytical methodologies for bacterial lipid analysis, with state-of-the-art techniques gaining wider adoption. MALDI-based MS analysis has revolutionized the field, enabling direct bacterial identification from complex biological samples without ex vivo growth, engineering of bacterial lipids for vaccine adjuvant development, and mapping of host-influenced lipid A modifications during Gram-negative bacterial infections. These developments have significantly advanced our understanding of host-pathogen interactions, particularly in relation to lipopolysaccharide (LPS) and its membrane anchor, lipid A.

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Lightning Talk
“A spatial multiomics workflow on a new benchtop MALDI-TOF instrument for deciphering the protein and lipid landscape in tissues”
Kate Stumpo, Ph.D.
Senior Marketing Manager, Bruker

Abstract: Spatial biology enables the visualization of different chemical landscapes in biological tissue making it an important technology in clinical research. MALDI mass spectrometry imaging
(MALDI Imaging) is well established for the spatial analysis of biomolecules in tissue. Additionally, multiomic spatial localization of lipids with expressed protein distributions can be
done which increases understanding of localized cellular processes in tissue. Here, we demonstrate an automated workflow on the neofleX, a new benchtop axial TOF.
FFPE sections of a human colorectal cancer sample were prepared for lipid imaging on the neofleX at 20 µm pixel size. To assess the spatial protein landscape, the same slide was
processed with the MALDI HiPLEX-IHC workflow, using 14 antibodies with photocleavable mass tags from AmberGen, Inc. The expressed protein distribution images of the mass tags from all of the 14 antibodies were obtained by automatic generation of ome.tiff files from a target list of the antibody mass tags as [M+H] + ion species and were visualized in the software SCiLS Scope. SCiLS Scope is a new tool for fast browsing through the color-coded ion images of the MALDI HiPLEX-IHC data, capable of visualizing both individual and composition ion images.
Protein expression and lipid imaging data were combined and mapped for examination of feature colocalization. Lipids colocalizing with individual tumor or immune markers from the
HiPLEX-IHC experiment were thereby obtained using the mapped images. For example, PC_32:0 was detected with higher abundance in the same region, where also cytokeratin marker PanCK was binding.
Our workflow provides a simple method to visualize the targeted spatial protein expression profiles from FFPE tissue. As an add on, our method can be combined with lipid imaging from the same tissue section to receive deeper insights into the cellular mechanisms of disease.

Speaker: Akos Vertes, The George Washington University

Topic: Spatial Metabolomics of Single Cells in their Natural Environment

Date: February 3, 2025

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 Dingyin Tao (owendtao@gmail.com) by Friday, January 31 if you will be attending the dinner.

Abstract: In multicellular organisms, cells assemble into tissues with specific functions. Tissue embedded cells operate a selection of metabolic pathways for the synthesis and degradation of a collection of small molecules that serve growth, signaling, and reproduction. Capturing the spatiotemporal distributions of metabolites, including lipids, with cellular granularity gives new insight into the functioning of tissues. We have developed ambient ionization techniques that can report on the metabolite content of functioning cells with high throughput and targeting capabilities. Image analysis and morphometry of brightfield and fluorescence microscope images is used to target selected cell types, followed by mid-IR laser ablation of individual cells. The ablation plume is ionized by an electrospray (laser ablation electrospray ionization, LAESI). Ion mobility separation (IMS) of the produced ions is followed by time-of-flight or Fourier transform ion cyclotron resonance mass spectrometry (MS) for the determination of cellular metabolite abundances. Cell-type specific small molecule compositions are determined and correlated with active metabolic pathways characteristic to cellular functions. Metabolite abundance distributions reflect population heterogeneity through metabolic noise levels and reveal hidden cellular phenotypes segregated into subpopulations functioning in specific metabolic states. Examples of spatial metabolomics are presented for various cell types including human hepatocytes, Arabidopsis thaliana and Allium cepa epidermal cells, and root nodule cells of Glycine max in nitrogen fixing symbiosis with Bradyrhizobium japonicum. In addition, using MS imaging (MSI) based on MALDI and laser desorption ionization (LDI) from silicon nanopost array (NAPA) nanophotonic platforms, the presence of a wide array of cyclic oligohexoses was discovered in the root nodules with degrees of polymerization in the 2 ≤ n ≤ 14 residue range. The spatial distributions of cyclic oligohexoses in the G. max nodules were established by NAPA-LDI-MSI. They appeared more concentrated at the edge of the infection zone or inner cortex and in the stem. At some locations, possibly in the vascular bundles surrounding the nodule and traversing the stem, the cyclic oligohexoses were especially abundant. At the same time, acyclic oligohexoses with 2 ≤ n ≤ 10 were also detected, and their distributions in the nodule sections were mapped. These linear or branching molecules were abundant in the infection zone, where the cyclic oligohexoses were less concentrated or absent.