Toward a Universal Sample Preparation Method for Denaturing Top-Down Proteomics of Complex Proteomes

A universal and standardized
sample preparation method becomes
vital for denaturing top-down proteomics (dTDP) to advance the scale
and accuracy of proteoform delineation in complex biological systems.
It needs to have high protein recovery, minimum bias, good reproducibility,
and compatibility with downstream mass spectrometry (MS) analysis.
Here, we employed a lysis buffer containing sodium dodecyl sulfate
for extracting proteoforms from cells and, for the first time, compared
membrane ultrafiltration (MU), chloroform–methanol precipitation
(CMP), and single-spot solid-phase sample preparation using magnetic
beads (SP3) for proteoform cleanup for dTDP. The MU method outperformed
CMP and SP3 methods, resulting in high and reproducible protein recovery
from both Escherichia coli cell (59
± 3%) and human HepG2 cell (86 ± 5%) samples without a significant
bias. Single-shot capillary zone electrophoresis (CZE)-MS/MS analyses
of the prepared E. coli and HepG2 cell
samples using the MU method identified 821 and 516 proteoforms, respectively.
Nearly 30 and 50% of the identified E. coli and HepG2 proteins are membrane proteins. CZE-MS/MS identified 94
histone proteoforms from the HepG2 sample with various post-translational
modifications, including acetylation, methylation, and phosphorylation.
Our results suggest that combining the SDS-based protein extraction
and the MU-based protein cleanup could be a universal sample preparation
method for dTDP. The MS raw data have been deposited to the ProteomeXchange
Consortium with the data set identifier PXD018248.