Identification of proteins in single-cell capillary electrophoresis fingerprints based on comigration with standard proteins.

Abstract

In the previous paper in this Journal, we reported the use of capillary sieving electrophoresis to characterize proteins expressed by single cancer cells at specific phases in the cell cycle. Analysis of the data revealed one component with cell cycle-dependent changes in expression at the 99% confidence limit. However, the amount of protein present in a single cell is far too small to allow its direct identification by mass spectrometry. In this paper, we report a method by which such proteins can be tentatively identified. We perform standard SDS-PAGE electrophoresis of the proteins contained within a homogenate prepared from an HT29 cell culture. Proteins extracted from bands in the gel are identified by mass spectrometry. The proteins also provide a set of standards that can be used to spike the sample before capillary sieving electrophoresis (CSE) separation; comigration is taken as evidence for the identity of the target protein. In a proof-of-principle experiment, a single band migrating at approximately 47 kDa was isolated from the SDS-PAGE gel generated from the HT29 cell line. Proteins extracted from this band were used to spike a CSE separation of the same extract. This band comigrated with a cell cycle-dependent component identified from single-cell analysis. In-gel digestion and LC/MS/MS were used to identify five proteins, including cytokeratin 18, which is the product of the most highly expressed gene in this cell line.