Single-cell differential detergent fractionation for detection of cytokeratin 8 proteoforms.

Simultaneous profiling of proteoforms and nucleic acids at the single-cell level, i.e., multi omics, directly links the central dogma. However, current single-cell approaches are limited in their ability to identify proteoforms while preserving the nucleus for further analysis. This limitation is especially pronounced in proteins where their proteoforms present diverse biological functions such as cytokeratin 8 (CK8), which, while commonly known for its structural role, is also involved in several diseases. Here, we present a single-cell western blot (scWB) integrated with differential detergent fractionation (DDF) to selectively solubilize and separate CK8 proteoforms while preserving nuclear integrity for subsequent nucleus-based assays. We report on assay development, including screening a panel of lysis buffers based on nonionic detergents and electrophoresis conditions to achieve a separation resolution between two proteoforms of up to 0.94 with an electric field of 30 V/cm, while preserving an intact nucleus. The cytoplasm-specific lysis approach (DDF buffer) demonstrated comparable solubilization efficiency to whole-cell solubilization (RIPA buffer), achieving proteoform solubilization in 14.3% and 10.3% of solubilized cells using DDF and RIPA buffers, respectively, while keeping the nucleus intact. To understand the broad applicability of the assay conditions, we scrutinized electrophoresis performance for resolving CK8 proteoforms across a panel of widely used breast cancer cell lines (MCF7, SKBR3, and MDA-MB-231), showing presence of proteoforms only in MCF7. Our approach allows for tailored solubilization, achieving reliable proteoform detection and nuclear retention across different cell types. Proteoform profiling at the single-cell level forms a basis for the exploration of the role of specific CK8 molecular forms in cellular processes.