Formaldehyde fixation helps preserving proteome state during single-cell proteomics sample processing and analysis

ABSTRACT: While mass spectrometry-based single cell proteomics (SCP) is gaining significant momentum, it is largely limited to a few laboratories worldwide. The ability to send samples to specialized core facilities, collaborators, would greatly benefit non-specialists laboratories or those unable to afford the costly instrumentation necessary to perform SCP analysis, and would help to popularize the use of the technology for biological applications. However, no methods have been tested in SCP which allow to “freeze” the proteome state while maintaining cell integrity to enable transfer of single cells suspensions between laboratories and/or prolonged sorting periods using fluorescence-activated cell sorting (FACS). Here we evaluate whether the widespread formaldehyde fixation could be used to maintain cell states enabling shipping between laboratories and on-site sorting, sample processing and mass spectrometry analysis. We demonstrate that short-term fixation using formaldehyde (FA) does not majorly affect protein recovery even in the absence of heating and strong detergent in single-cell proteomics and One-Tip analyses and allow maintaining analytical depth in comparison to classical workflows without fixation. Additionally, we show that fixation preserves drug-induced specific perturbations of protein abundance during cell sorting and sample preparation for SCP analysis. Our study has implication in single-cell and sensitive proteomics and would help provide biologists and other non-specialist researcher access to the technology, while also enables intracellular labelling using antibodies for FACS. This also helps the field expand toward multidimensional analysis where fixing the proteome state at a certain time such as for certain dynamic PTMs is crucial.

INSTRUMENT(S): Orbitrap Astral

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Pierre Sabatier  

LAB HEAD: Jesper V. Olsen

PROVIDER: PXD054445 | Pride | 2025-02-05

REPOSITORIES: pride