Top-Down Proteomics at High Magnetic Field: Posttranslational Modifications and High Molecular Weight Proteins from M-Phase Arrested HeLa Cells

摘要

Proteins up to 50 kDa are routinely baseline resolved for accurate precursor mass determination. Fragment ions are generated with nozzle-skimmer dissociation to identify proteins (greater than 30 kDa). All separations are performed with nano-LC instead of the traditional larger-bore columns associated with previous reports. Analysis of two M-phase arrested HeLa cell preparations resulted in identification of previously described proteins that exhibit up-regulated phosphorylation under this treatment. Currently, 23 phosphorylated proteins have been identified. Because of the nature of the data-dependent MS/MS experiment, multiple injections per fraction increases the dynamic range, number of identified proteins, phosphoproteins, and other PTMs (acetylation, methylation, etc.).