Biology subjects

Woessmann, J.

Publications and source records attributed to Woessmann, J..

3 recordsLinked to original sources

bioRxiv2024

Quantitative Label-Free Single-Cell Proteomics on the Orbitrap Astral MS

Single-cell proteomics by mass spectrometry (scp-MS) holds the potential to provide unprecedented insights into molecular features directly linked to the cellular phenotype, while deconvoluting complex organisms into their basic building blocks. Tailored sample preparation that maximizes the extracted amount of material that is introduced into the mass spectrometer has rapidly propelled the field forward. However, the measured signal is still at the lower edge of detection approaching the sensitivity boundary of current instrumentation. Here, we investigate the capacity of the enhanced sensitivity of the Orbitrap Astral mass spectrometer to facilitate deeper proteome profiles from low-input to single-cell samples. We carry out a comprehensive data acquisition method survey to pinpoint which parameters provide most sensitivity. Furthermore, we explore the quantitative accuracy of the obtained measurements to ensure that the obtained abundances are in line with expected ground truth values. We culminate our technical exploration by generating small datasets from two cultured cell lines and a primary bone marrow sample, to showcase obtainable proteome coverage differences from different source materials. Finally, as a proof of concept we explore protein covariation to showcase how information on known protein complexes is captured inherently in our scp-MS data.

biochemistry↗

bioRxiv2024

Refining Spatial Proteomics by Mass Spectrometry: An Efficient Workflow Tailored for Archival Tissue

BackgroundFormalin-fixed, paraffin-embedded (FFPE) tissue remains the gold standard for extensively archiving biological specimens, providing biobanks with large repositories of retrospective potential. However, while formalin crosslinking is effective at preserving tissue, it poses significant challenges for extracting molecular information, including the proteome. Traditionally, this process required high levels of input material, which, in turn, limited the ability to preserve cell-type heterogeneity and spatial information. To address these limitations, we developed an easily adaptable and highly efficient workflow for extracting deep proteomes from low-input materials, such as biopsies used in routine histopathological diagnostics. MethodsWe compared the extraction efficiency of pancreatic acinar cells identified in FFPE tissue samples stained with conventional hematoxylin-eosin (H&E) against that of cells isolated from tissue samples immunostained for the epithelial cell adhesion molecule (EpCAM) across material inputs ranging from 1,166 to 800,000 {micro}m2 (estimated to 2 to 1,310 cells in volume). Cells were isolated using laser capture microdissection and subsequently analyzed using Liquid Chromatography-Tandem Mass Spectrometry. ResultsSimilar yields for both methods were observed, with EpCAM-positive cells yielding slightly higher results--approximately 1,200 unique protein groups at the lowest input and up to 5,900 at the highest. In cells isolated from H&E-stained tissue, [~]900 to [~]5,200 protein groups were identified. We decided that the optimal balance for our workflow, ensuring maximum protein identification while minimizing input material, lies within the range of approximately 50,000 to 100,000 {micro}m2. With these results, we tested spatial capabilities and biological relevance by isolating cancer cells from biopsies of pancreatic cancer, lung cancer, or glioblastoma, with the first two being stained with EpCAM and the latter being stained against the tumor-suppressor protein p53. We successfully identified tissue-specific protein expressions and observed prominent clustering of all cell populations. DiscussionOur results highlight the feasibility of performing spatial proteomics on FFPE tissue using minimal input material. This adaptable methodology opens up possibilities for investigating cell-type-specific biology while preserving spatial and histological information.

bioengineering↗

bioRxiv2023

Assessing the role of trypsin in quantitative plasma- and single-cell proteomics towards clinical application

Mass spectrometry-based bottom-up proteomics is rapidly evolving and routinely applied in large biomedical studies. Proteases are a central component of every bottom-up proteomics experiment, digesting proteins into peptides. Trypsin has been the most widely applied protease in proteomics, due to its characteristics. With ever-larger cohort sizes and possible future clinical application of mass spectrometry-based proteomics, the technical impact of trypsin becomes increasingly relevant. To assess possible biases introduced by trypsin digestion, we evaluated the impact of eight commercially available trypsins in a variety of bottom-up proteomics experiments, and across a range of protease concentrations and storage times. To investigate the universal impact of these technical attributes, we included bulk HeLa-cell lysate, human plasma and single HEK293 cells, which were analyzed over a range of Selected Reaction Monitoring (SRM), Data-Independent Acquisition (DIA), and Data-Dependent Acquisition (DDA) instrument methods on three LC-MS instruments. Quantification methods employed encompassed both label-free approaches and absolute quantification utilizing spike-in heavy-labeled recombinant protein fragment standards. Based on this extensive dataset, we report variations between commercial trypsins, their source, as well as their concentration. Furthermore, we provide suggestions on the handling of trypsin in large scale studies.

molecular biology↗