Biology subjects

Ostergaard, O.

Publications and source records attributed to Ostergaard, O..

3 recordsLinked to original sources

bioRxiv2026

Comparison of extracellular vesicles and mechanically induced vesicles for structure determination of membrane proteins

The structural and functional characteristics of membrane proteins can be influenced by the composition of the membrane. Consequently, native membranes are most relevant for the study of receptors and other membrane proteins. In this study, we investigated two types of cell-derived vesicles: natively shed extracellular vesicles (EVs) and mechanically derived vesicles (MVs). To this end, we utilized the human breast cancer cell line SKBR3, which strongly overexpresses the receptor HER2. We designed a protocol based on designed ankyrin repeat proteins (DARPins) to purify EVs and MVs enriched in HER2, and to ensure the native orientation of the HER2 receptors within the vesicle. The isolated HER2-containing EVs and MVs were characterized by cryo-EM, cryo-electron tomography (cryo-ET) and mass spectrometry (MS), which revealed fundamental differences between the different vesicle types. Our study highlights the greater structural diversity of EVs over MVs. A single particle cryo-EM analysis and classification of all visible receptors on the vesicle surface yielded electron density consistent with HER2 at modest resolution. Taken together, our results suggest that MVs can serve better than EVs as a suitable platform for the structure determination of membrane proteins within their native membrane environments.

biochemistry↗

bioRxiv2025

Benchmarking enrichment and depletion methods for quantitative plasma proteomics in different plasma types and the correlation to clinical routine assays.

Plasma proteomics based on mass spectrometry has great potential for biomarker discovery. Plasma is challenging for mass spectrometry due to high dynamic range in protein abundance. Several workflows have been developed to overcome this, and in this study, we compare prominent workflows using platelet-poor-(PPP), platelet-rich plasma (PRP) and serum (SER). Our results show that depletion workflows including Top14 depletion and acid precipitation allow quantification of very different proteomes than methods based on enrichments of extracellular vesicles such as bead-based enrichment or ultracentrifugation. Enrichment methods are superior in terms of proteome depth and quantitative performance but may be less robust in large cohorts. There is a very high correlation between PPP and PRP samples with all methods and less to SER samples - especially with enrichment workflows. The correlation of 10 protein measurements, performed by clinical routine processes on a Cobas system, showed heterogeneous results. Low abundant proteins with biological dynamics within a healthy cohort, including c-reactive protein and lipoprotein(a), correlated very well to proteomic workflows while others, including albumin and transferrin, correlated poorly. In conclusion, the workflow for plasma proteomics should be aligned with the aim of the analysis and the setup of the sample collection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/686186v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@10b62a7org.highwire.dtl.DTLVardef@79d4f1org.highwire.dtl.DTLVardef@8b21a3org.highwire.dtl.DTLVardef@4d85d9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

biochemistry↗

bioRxiv2023

Narrow-window DIA: Ultra-fast quantitative analysis of comprehensive proteomes with high sequencing depth

Mass spectrometry (MS)-based proteomics aims to characterize comprehensive proteomes in a fast and reproducible manner. Here, we present an ultra-fast scanning data-independent acquisition (DIA) strategy consisting on 2-Th precursor isolation windows, dissolving the differences between data-dependent and independent methods. This is achieved by pairing a Quadrupole Orbitrap mass spectrometer with the asymmetric track lossless (Astral) analyzer that provides >200 Hz MS/MS scanning speed, high resolving power and sensitivity, as well as low ppm-mass accuracy. Narrow-window DIA enables profiling of up to 100 full yeast proteomes per day, or [~]10,000 human proteins in half-an-hour. Moreover, multi-shot acquisition of fractionated samples allows comprehensive coverage of human proteomes in [~]3h, showing comparable depth to next-generation RNA sequencing and with 10x higher throughput compared to current state-of-the-art MS. High quantitative precision and accuracy is demonstrated with high peptide coverage in a 3-species proteome mixture, quantifying 14,000+ proteins in a single run in half-an-hour. TeaserAccurate and precise label-free quantification with comprehensive proteome coverage using narrow-window DIA

systems biology↗