Biology subjects

Neumann, E. K.

Publications and source records attributed to Neumann, E. K..

4 recordsLinked to original sources

bioRxiv2021

Direct Imaging and Identification of Proteoforms up to 70 kDa from Human Tissue

Imaging of proteoforms in human tissues is hindered by low molecular specificity and limited proteome coverage. Here, we introduce proteoform imaging mass spectrometry (PiMS), which increases the size limit for proteoform detection and identification by 4-fold compared to reported methods, and reveals tissue localization of proteoforms at <80 m spatial resolution. PiMS advances proteoform imaging by combining ambient nanospray desorption electrospray ionization (nano-DESI) with ion detection using individual ion mass spectrometry (I2MS). We demonstrate the first proteoform imaging of human kidney, identifying 169 of 400 proteoforms <70 kDa using top-down mass spectrometry and database lookup from the human proteoform atlas, including dozens of key enzymes in primary metabolism. PiMS images reveal distinct spatial localizations of proteoforms to both anatomical structures and cellular neighborhoods in the vasculature, medulla, and cortex regions of the human kidney. The benefits of PiMS are poised to increase proteome coverage for label-free protein imaging of tissues. TeaserNano-DESI combined with individual ion mass spectrometry generates images of proteoforms up to 70 kDa.

biochemistry↗

bioRxiv2021

Optimized Workflow for High Spatial Resolution MALDI Imaging Mass Spectrometry ofFresh-Frozen Bone

Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis, osteoporosis, osteomyelitis, and cancer. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) enable the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of native osseous tissue for MALDI IMS has been difficult due to the mineralized composition and heterogenous morphology of the tissue, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs allowing the distribution of endogenous lipids to be linked to specific tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with a microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. Subliming matrix does not induce further bone marrow cracking, and recrystallizing the deposited matrix improves lipid signal. High spatial resolution (10 m) MALDI IMS was employed to characterize lipid distributions in fresh-frozen bone, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment.

biochemistry↗

bioRxiv2021

Autofluorescence microscopy as a label-free tool for renal histology and glomerular segmentation

Automated spatial segmentation models can enrich spatio-molecular omics analyses by providing a link to relevant biological structures. We developed segmentation models that use label-free autofluorescence (AF) microscopy to recognize multicellular functional tissue units (FTUs) (glomerulus, proximal tubule, descending thin limb, ascending thick limb, distal tubule, and collecting duct) and gross morphological structures (cortex, outer medulla, and inner medulla) in the human kidney. Annotations were curated using highly specific multiplex immunofluorescence and transferred to co-registered AF for model training. All FTUs (except the descending thin limb) and gross kidney morphology were segmented with high accuracy: >0.85 F1-score, and Dice-Sorensen coefficients >0.80, respectively. This workflow allowed lipids, profiled by imaging mass spectrometry, to be quantitatively associated with segmented FTUs. The segmentation masks were also used to acquire spatial transcriptomics data from collecting ducts. Consistent with previous literature, we demonstrated differing transcript expression of collecting ducts in the inner and outer medulla.

bioinformatics↗

bioRxiv2020

Highly Multiplexed Immunofluorescence of the Human Kidney using Co-Detection by Indexing (CODEX)

The human kidney is composed of many cell types that vary in their abundance and distribution from organ to organ. As these cell types perform unique and essential functions, it is important to confidently label each within a single tissue to more accurately assess tissue architecture. Towards this goal, we demonstrate the use of co-detection by indexing (CODEX) multiplexed immunofluorescence for visualizing 23 antigens within the human kidney. Using CODEX, many of the major cell types and substructures, such as collecting ducts, glomeruli, and thick ascending limb, were visualized within a single tissue section. Of these antibodies, 19 were conjugated in-house, demonstrating the flexibility and utility of this approach for studying the human kidney using traditional antibody markers. We performed a pilot study showing that the studied tissues had on average 84 {+/-} 11 cells per mm2 with the most variance seen within the cells containing vimentin and aquaporin 1, while cells containing -smooth muscle actin and CD31 possessed a high degree of uniformity between the samples. These precursory data show the power of CODEX multiplexed IF for surveying the cellular diversity of the human kidney and have potential applications within pathology, histology, and building anatomical atlases.

cell biology↗