Biology subjects

Üresin, N.

Publications and source records attributed to Üresin, N..

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

Unraveling the proteome landscape of mouse hematopoietic stem and progenitor compartment with high sensitivity low-input proteomics

Proteins play a key role in defining cellular phenotypes, yet comprehensive proteomic analysis often requires substantial input material, posing challenges in studying rare populations in complex cell systems. Here, we present an accessible, label-free low-input proteomics workflow that allows for comprehensive proteome coverage reminiscent of classical bulk samples from only 500 cells and showcase its application in murine hematopoiesis. With this approach, we construct a proteomic map of hematopoietic stem and progenitor cell (HSPC) populations isolated by fluorescence-activated cell sorting (FACS) from the bone marrow of a single mouse, identifying approximately 7,000 proteins per cell population. Our study recapitulates the differentiation trajectories along the megakaryocytic-erythroid and granulocytic-monocytic lineages. We specifically focus on the dynamics of transcriptional regulators and provide insights into both known and novel population-specific factors. Furthermore, we extend our exploration to the most primitive stem and progenitor compartment, and identify ADP-Ribosyltransferase ART4 (CD297) as a novel cell surface marker that can potentially be used to enrich for long-term hematopoietic stem cells (LT-HSC). The low-input proteomics workflow presented here holds promise for overcoming the challenges associated with analyzing proteomes of rare cell populations, thereby paving the way for broader applications in biomedical research.

cell biology↗

bioRxiv2021

Real-Time Search Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics

In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed single-cell proteomics requires high ion injection times and high-resolution spectra to quantify the single-cell signal, however the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and present a new acquisition strategy termed RETICLE (RTS Enhanced Quant of Single Cell Spectra) that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. We show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at a MS2 injection time of 750ms using a 2h gradient.

biochemistry↗