Limiting the impact of protein leakage in single-cell proteomics.

Limiting artifacts during sample preparation can significantly increase data quality in single-cell proteomics experiments. Towards this goal, we characterize the impact of protein leakage by analyzing thousands of primary single cells from mouse trachea. The cells were prepared either fresh immediately after dissociation or first cryopreserved and prepared at a later date. We directly identify permeabilized cells by imaging a cell permeable dye and use the data to define a signature for protein leakage. This signature is similar across diverse cell types and reflects increased leakage propensities for cytosolic and nuclear proteins compared to membrane and mitochondrial proteins. A classifier based on the signature allowed for the accurate identification of permeabilized cells across cell types and species. The classifier is integrated into QuantQC ( scp.slavovlab.net/QuantQC ) to support its application to diverse samples and workflows.