MassIVE MSV000080365

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

Description

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. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: top-down proteomics ; serial size exclusion chromatography ; large proteins

Contact

Principal Investigators:
(in alphabetical order)
Prof. Ying Ge
Submitting User: Ge_Lab_16

Publications

.
Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy.
Anal Chem. 2017 May 16;89(10):5467-5475. doi: 10.1021/acs.analchem.7b00380. Epub 2017 May 2.

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Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

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Number of distinct proteins quantified across all analyses (original submission and reanalyses) associated with this dataset.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

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Number of distinct proteins found to be differentially abundant in at least one comparison across all analyses (original submission and reanalyses) associated with this dataset.

A protein is differentially abundant if its change in abundance across conditions is found to be statistically significant with an adjusted p-value <= 0.05 and lists no issues associated with statistical tests for differential abundance.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
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