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Proteomes 2017, 5(2), 13; doi:10.3390/proteomes5020013

A Routine ‘Top-Down’ Approach to Analysis of the Human Serum Proteome

1
Department of Molecular Physiology, The Molecular Medicine Research Group, School of Medicine, Western Sydney University, Campbelltown, NSW 2150, Australia
2
Department of High Risk Obstetrics, RPA Women and Babies, Royal Prince Alfred Hospital, Sydney, NSW 2050, Australia
3
Faculty of Graduate Studies, and the Departments of Health Sciences and Biological Sciences, Brock University, St. Catharines, ON L2S 3A1, Canada
*
Author to whom correspondence should be addressed.
Academic Editor: Jacek R. Wisniewski
Received: 10 March 2017 / Revised: 30 May 2017 / Accepted: 30 May 2017 / Published: 6 June 2017
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Abstract

Serum provides a rich source of potential biomarker proteoforms. One of the major obstacles in analysing serum proteomes is detecting lower abundance proteins owing to the presence of hyper-abundant species (e.g., serum albumin and immunoglobulins). Although depletion methods have been used to address this, these can lead to the concomitant removal of non-targeted protein species, and thus raise issues of specificity, reproducibility, and the capacity for meaningful quantitative analyses. Altering the native stoichiometry of the proteome components may thus yield a more complex series of issues than dealing directly with the inherent complexity of the sample. Hence, here we targeted method refinements so as to ensure optimum resolution of serum proteomes via a top down two-dimensional gel electrophoresis (2DE) approach that enables the routine assessment of proteoforms and is fully compatible with subsequent mass spectrometric analyses. Testing included various fractionation and non-fractionation approaches. The data show that resolving 500 µg protein on 17 cm 3–10 non-linear immobilised pH gradient strips in the first dimension followed by second dimension resolution on 7–20% gradient gels with a combination of lithium dodecyl sulfate (LDS) and sodium dodecyl sulfate (SDS) detergents markedly improves the resolution and detection of proteoforms in serum. In addition, well established third dimension electrophoretic separations in combination with deep imaging further contributed to the best available resolution, detection, and thus quantitative top-down analysis of serum proteomes. View Full-Text
Keywords: deep Imaging; Lithium Dodecyl Sulfate; prefractionation; postfractionation; proteomics; proteoforms; three-dimensional gel electrophoresis; two-dimensional gel electrophoresis deep Imaging; Lithium Dodecyl Sulfate; prefractionation; postfractionation; proteomics; proteoforms; three-dimensional gel electrophoresis; two-dimensional gel electrophoresis
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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D’Silva, A.M.; Hyett, J.A.; Coorssen, J.R. A Routine ‘Top-Down’ Approach to Analysis of the Human Serum Proteome. Proteomes 2017, 5, 13.

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