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Authors Cai W, Tucholski T, Chen B, Alpert AJ, McIlwain S, Kohmoto T, Jin S, Ge Y
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Journal Anal. Chem. Volume: 89 Issue: 10 Pages: 5467-5475
Publish Date 2017 May 16
PubMed ID 28406609
PMC ID 5511113
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 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.

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