Dataset Information

Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation.

ABSTRACT: Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10-fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable-core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease.

SUBMITTER: Brunner AD

PROVIDER: S-EPMC8884154 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation.

Brunner Andreas-David AD Thielert Marvin M Vasilopoulou Catherine C Ammar Constantin C Coscia Fabian F Mund Andreas A Hoerning Ole B OB Bache Nicolai N Apalategui Amalia A Lubeck Markus M Richter Sabrina S Fischer David S DS Raether Oliver O Park Melvin A MA Meier Florian F Theis Fabian J FJ Mann Matthias M

Molecular systems biology 20220301 3

Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more ...[more]

PMID: 35226415

Similar Datasets

Project description:BackgroundQuantification of drug-target binding is critical for confirming that drugs reach their intended protein targets, understanding the mechanism of action, and interpreting dose-response relationships. For covalent inhibitors, target engagement can be inferred by free target levels before and after treatment. Targeted mass spectrometry assays offer precise protein quantification in complex biological samples and have been routinely applied in pre-clinical studies to quantify target engagement in frozen tumor tissues for oncology drug development. However, frozen tissues are often not available from clinical trials so it is critical that assays are applicable to formalin-fixed, paraffin-embedded (FFPE) tissues in order to extend mass spectrometry-based target engagement studies into clinical settings.MethodsWild-type RAS and RASG12C was quantified in FFPE tissues by a highly optimized targeted mass spectrometry assay that couples high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) with internal standards. In a subset of samples, technical reproducibility was evaluated by analyzing consecutive tissue sections from the same tumor block and biological variation was accessed among adjacent tumor regions in the same tissue section.ResultsWild-type RAS protein was measured in 32 clinical non-small cell lung cancer tumors (622-2525 amol/µg) as measured by FAIMS-PRM mass spectrometry. Tumors with a known KRASG12C mutation (n = 17) expressed a wide range of RASG12C mutant protein (127-2012 amol/µg). The variation in wild-type RAS and RASG12C measurements ranged 0-18% CV across consecutive tissue sections and 5-20% CV among adjacent tissue regions. Quantitative target engagement was then demonstrated in FFPE tissues from 2 xenograft models (MIA PaCa-2 and NCI-H2122) treated with a RASG12C inhibitor (AZD4625).ConclusionsThis work illustrates the potential to expand mass spectrometry-based proteomics in preclinical and clinical oncology drug development through analysis of FFPE tumor biopsies.

Dataset Information

Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation.

Publications

Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets