Enhanced feature matching in single-cell proteomics characterizes response to IFN-γ and reveals co-existence of different cell states-Bulk Proteomics experiment

ABSTRACT: Proteome analysis by data-independent acquisition (DIA) has become a powerful approach to obtain deep proteome coverage, and has gained recent traction for label-free analysis of single cells. However, optimal experimental design for DIA-based single-cell proteomics has not been fully explored, and performance metrics of subsequent data analysis tools remain to be evaluated. Therefore, we here present DIA-ME, a data analysis strategy that exploits the co-analysis of low-input samples with a so-called matching enhancer (ME) of higher input, to increase sensitivity, proteome coverage, and data completeness. We evaluate the matching specificity of DIA-ME by a two-proteome model, and demonstrate that false discovery and false transfers are maintained at low levels when using DIA-NN software, while preserving quantification accuracy. We apply DIA-ME to investigate the proteome response of U-2 OS cells to interferon gamma (IFN-γ) in single cells, and recapitulate the time-resolved induction of IFN-γ response proteins as observed in bulk material. Moreover, we observe co- and anti-correlating patterns of protein expression within the same cell, indicating mutually exclusive protein modules and the co-existence of different cell states. Collectively our data show that DIA-ME is a powerful, scalable, and easy-to-implement strategy for single-cell proteomics.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Osteosarcoma

SUBMITTER: Syed Azmal Ali  

LAB HEAD: Prof. Jeroen Krijgsveld

PROVIDER: PXD048162 | Pride | 2024-08-22

REPOSITORIES: Pride