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Issue 23, 2019
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An SDS-PAGE based proteomic approach for N-terminome profiling

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Abstract

Initial sample quantity, solubilization, separation, and visualization of proteins or their proteolytically altered products are some of the challenges of the currently available solution-based N-termini enrichment methods. We therefore took advantage of the conventional SDS-PAGE system and attempted to address these challenges by proposing a simple yet reproducible, negative selection N-termini enrichment strategy coupled with mass spectrometry based protein identification. It includes in-gel protein level labeling of primary amines using d6-acetic anhydride and post-digestion negative selection of labeled N-terminal peptide(s) using N-hydroxysuccinimide activated agarose beads. We demonstrated the superiority of our method by successfully enriching protein N-termini from as low as 10 ng of bovine serum albumin. The method was validated for its applicability to a complex mixture of proteins by selectively enriching neo-N-termini generated by a site specific protease Glu-C. Its effectiveness for deep N-terminome profiling was also shown using human cell lysate. In addition, a system-wide label-free quantitative proteomic analysis of N-termini in MMP2-perturbed HCT8 cell secretome revealed substrates of several extra- and intra-cellular proteases, which are part of cell growth and proliferation and degradation pathways. In brief, the proposed method demonstrates an effective strategy not only to detect N-termini from a single protein but also for the deep and quantitative analysis of N-terminome from a limited sample amount.

Graphical abstract: An SDS-PAGE based proteomic approach for N-terminome profiling

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Publication details

The article was received on 22 Aug 2019, accepted on 24 Sep 2019 and first published on 01 Oct 2019


Article type: Paper
DOI: 10.1039/C9AN01616C
Analyst, 2019,144, 7001-7009

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    An SDS-PAGE based proteomic approach for N-terminome profiling

    P. Kaushal, Y. Kwon, S. Ju and C. Lee, Analyst, 2019, 144, 7001
    DOI: 10.1039/C9AN01616C

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