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  1. Article ; Online: Assessing the Role of Trypsin in Quantitative Plasma and Single-Cell Proteomics toward Clinical Application

    Woessmann, Jakob / Petrosius, Valdemaras / Üresin, Nil / Kotol, David / Aragon-Fernandez, Pedro / Hober, Andreas / auf dem Keller, Ulrich / Edfors, Fredrik / Schoof, Erwin M.

    Analytical Chemistry. 2023 Aug. 28, v. 95, no. 36 p.13649-13658

    2023  

    Abstract: Mass spectrometry-based bottom-up proteomics is rapidly evolving and routinely applied in large-scale biomedical studies. Proteases are a central component of every bottom-up proteomics experiment, digesting proteins into peptides. Trypsin has been the ... ...

    Abstract Mass spectrometry-based bottom-up proteomics is rapidly evolving and routinely applied in large-scale 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. The quantification methods employed encompassed both label-free approaches and absolute quantification utilizing spike-in heavy-labeled recombinant protein fragment standards. Based on this extensive data set, we report variations between commercial trypsins, their source, and their concentration. Furthermore, we provide suggestions on the handling of trypsin in large-scale studies.
    Keywords analytical chemistry ; data collection ; digestion ; human cell lines ; humans ; mass spectrometry ; peptides ; proteomics ; recombinant proteins ; trypsin
    Language English
    Dates of publication 2023-0828
    Size p. 13649-13658.
    Publishing place American Chemical Society
    Document type Article ; Online
    ZDB-ID 1508-8
    ISSN 1520-6882 ; 0003-2700
    ISSN (online) 1520-6882
    ISSN 0003-2700
    DOI 10.1021/acs.analchem.3c02543
    Database NAL-Catalogue (AGRICOLA)

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  2. Article ; Online: Assessing the Role of Trypsin in Quantitative Plasma and Single-Cell Proteomics toward Clinical Application.

    Woessmann, Jakob / Petrosius, Valdemaras / Üresin, Nil / Kotol, David / Aragon-Fernandez, Pedro / Hober, Andreas / Auf dem Keller, Ulrich / Edfors, Fredrik / Schoof, Erwin M

    Analytical chemistry

    2023  Volume 95, Issue 36, Page(s) 13649–13658

    Abstract: Mass spectrometry-based bottom-up proteomics is rapidly evolving and routinely applied in large-scale biomedical studies. Proteases are a central component of every bottom-up proteomics experiment, digesting proteins into peptides. Trypsin has been the ... ...

    Abstract Mass spectrometry-based bottom-up proteomics is rapidly evolving and routinely applied in large-scale 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. The quantification methods employed encompassed both label-free approaches and absolute quantification utilizing spike-in heavy-labeled recombinant protein fragment standards. Based on this extensive data set, we report variations between commercial trypsins, their source, and their concentration. Furthermore, we provide suggestions on the handling of trypsin in large-scale studies.
    MeSH term(s) Humans ; Trypsin ; HEK293 Cells ; HeLa Cells ; Proteomics ; Peptide Hydrolases
    Chemical Substances Trypsin (EC 3.4.21.4) ; Peptide Hydrolases (EC 3.4.-)
    Language English
    Publishing date 2023-08-28
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1508-8
    ISSN 1520-6882 ; 0003-2700
    ISSN (online) 1520-6882
    ISSN 0003-2700
    DOI 10.1021/acs.analchem.3c02543
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

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  3. Article ; Online: Exploration of cell state heterogeneity using single-cell proteomics through sensitivity-tailored data-independent acquisition.

    Petrosius, Valdemaras / Aragon-Fernandez, Pedro / Üresin, Nil / Kovacs, Gergo / Phlairaharn, Teeradon / Furtwängler, Benjamin / Op De Beeck, Jeff / Skovbakke, Sarah L / Goletz, Steffen / Thomsen, Simon Francis / Keller, Ulrich Auf dem / Natarajan, Kedar N / Porse, Bo T / Schoof, Erwin M

    Nature communications

    2023  Volume 14, Issue 1, Page(s) 5910

    Abstract: Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount of material encapsulated ... ...

    Abstract Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount of material encapsulated in a single cell however, raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, single-cell proteomics by Mass Spectrometry (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, although further development is needed to realize its full potential. To this end, we carry out comprehensive analysis of orbitrap-based data-independent acquisition (DIA) for limited material proteomics. Notably, we find a fundamental difference between optimal DIA methods for high- and low-load samples. We further improve our low-input DIA method by relying on high-resolution MS1 quantification, thus enhancing sensitivity by more efficiently utilizing available mass analyzer time. With our ultra-low input tailored DIA method, we are able to accommodate long injection times and high resolution, while keeping the scan cycle time low enough to ensure robust quantification. Finally, we demonstrate the capability of our approach by profiling mouse embryonic stem cell culture conditions, showcasing heterogeneity in global proteomes and highlighting distinct differences in key metabolic enzyme expression in distinct cell subclusters.
    MeSH term(s) Animals ; Mice ; Proteomics ; Mass Spectrometry ; Mouse Embryonic Stem Cells ; Proteome ; Single-Cell Analysis
    Chemical Substances Proteome
    Language English
    Publishing date 2023-09-22
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-023-41602-1
    Database MEDical Literature Analysis and Retrieval System OnLINE

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