Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy

Mass
spectrometry (MS)-based top-down proteomics is a powerful
method for the comprehensive analysis of proteoforms that arise from
genetic variations and post-translational modifications (PTMs). However,
top-down MS analysis of high molecular weight (MW) proteins remains
challenging mainly due to the exponential decay of signal-to-noise
ratio with increasing MW. Size exclusion chromatography (SEC) is a
favored method for size-based separation of biomacromolecules but
typically suffers from low resolution. Herein, we developed a serial
size exclusion chromatography (sSEC) strategy to enable high-resolution
size-based fractionation of intact proteins (10–223 kDa) from
complex protein mixtures. The sSEC fractions could be further separated
by reverse phase chromatography (RPC) coupled online with high-resolution
MS. We have shown that two-dimensional (2D) sSEC-RPC allowed for the
identification of 4044 more unique proteoforms and a 15-fold increase
in the detection of proteins above 60 kDa, compared to one-dimensional
(1D) RPC. Notably, effective sSEC-RPC separation of proteins significantly
enhanced the detection of high MW proteins up to 223 kDa and also
revealed low abundance proteoforms that are post-translationally modified.
This sSEC method is MS-friendly, robust, and reproducible and, thus,
can be applied to both high-efficiency protein purification and large-scale
proteomics analysis of cell or tissue lysate for enhanced proteome
coverage, particularly for low abundance and high MW proteoforms.