Informed Data-Independent Acquisition Enables Targeted Quantification of Key Regulatory Proteins in Cell Fate Decision at Single-Cell Resolution
Woessmann, J.; Petrosius, V.; Schovsbo, S.; Arrey, T. N.; Furtwaengler, B.; Op de Beeck, J.; Damoc, E.; Porse, B. T.; Schoof, E. M.
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Cellular differentiation processes are largely orchestrated by variation in transcription factor (TF) abundance. Since these proteins are usually expressed at extremely low levels, studying TF-driven cellular processes using single-cell proteomics by mass spectrometry (scp-MS) has been challenging. Here we present informed DIA (iDIA), an acquisition method tailored towards low-input and scp-MS. iDIA combines targeted and global proteomics in a single acquisition scheme and is by design universally applicable on different MS instrumentation. By combining wide window (ww)PRM scans with DIA acquisition, we observed a median 4-fold improvement in limit of quantification with targeted acquisitions, while maintaining biologically meaningful global proteome coverage. We utilize this gained sensitivity to quantify TFs in single murine hematopoietic stem and progenitor cells and showcase their lineage-specific regulation.
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