Biology subjects

Petzoldt, J.

Publications and source records attributed to Petzoldt, J..

4 recordsLinked to original sources

bioRxiv2023

Evaluating the capabilities of the Astral mass analyzer for single-cell proteomics

The complexity of human physiology arises from well-orchestrated interactions between trillions of single cells in the body. While single-cell RNA sequencing (scRNA-seq) has enhanced our understanding of cell diversity, gene expression alone does not fully characterize cell phenotypes. Additional molecular dimensions, such as proteins, are needed to define cellular states accurately. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool for comprehensive protein analysis, including single-cell applications. However, challenges remain in terms of throughput and proteomic depth, in order to maximize the biological impact of single-cell proteomics by Mass Spectrometry (scp-MS) workflows. This study leverages a novel high-resolution, accurate mass (HRAM) instrument platform, consisting of both an Orbitrap and an innovative HRAM Asymmetric Track Lossless (Astral) analyzer. The Astral analyzer offers high sensitivity and resolution through lossless ion transfer and a unique flight track design. We evaluate the performance of the Thermo Scientific Orbitrap Astral MS using Data-Independent Acquisition (DIA) and assess proteome depth and quantitative precision for ultra-low input samples. Optimal DIA method parameters for single-cell proteomics are identified, and we demonstrate the ability of the instrument to study cell cycle dynamics in Human Embryonic Kidney (HEK293) cells, and cancer cell heterogeneity in a primary Acute Myeloid Leukemia (AML) culture model.

systems biology↗

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↗

bioRxiv2023

Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High-Throughput Quantitative Analysis.

The growing trend towards high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time, and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass resolving quadrupole, the Orbitrap and the novel Asymmetric Track Lossless (Astral) analyzer. The new hybrid instrument enables faster acquisition of high-resolution accurate mass (HRAM) MS/MS spectra compared to state-of-the-art mass spectrometers. Accordingly, new proteomics methods were developed that leverage the strengths of each HRAM analyzer, whereby the Orbitrap analyzer performs full scans with high dynamic range and resolution, synchronized with Astral analyzers acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/543408v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1eedfdeorg.highwire.dtl.DTLVardef@1ab7939org.highwire.dtl.DTLVardef@a3b2f8org.highwire.dtl.DTLVardef@13ac46b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Evaluating the performance of the Astral mass analyzer for quantitative proteomics using data independent acquisition

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high quality quantitative measurements across a wide dynamic range. We also use a newly developed extra-cellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5,000 plasma proteins in a 60-minute gradient with the Orbitrap Astral mass spectrometer.

biochemistry↗