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

ABSTRACT: 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 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 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, especially for low abundance and high MW proteoforms.

INSTRUMENT(S): maXis

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Prof. Ying Ge 

PROVIDER: MSV000080365 | MassIVE | Tue Nov 29 12:48:00 GMT 2016

REPOSITORIES: MassIVE