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Proteomes, Volume 5, Issue 2 (June 2017)

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Editorial

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Open AccessEditorial Clinical Proteomics: From Biological Sample to Clinical Exploitation
Proteomes 2017, 5(2), 10; doi:10.3390/proteomes5020010
Received: 5 April 2017 / Revised: 5 April 2017 / Accepted: 5 April 2017 / Published: 6 April 2017
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(This article belongs to the Special Issue Clinical Proteomics)

Research

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Open AccessArticle A Routine ‘Top-Down’ Approach to Analysis of the Human Serum Proteome
Proteomes 2017, 5(2), 13; doi:10.3390/proteomes5020013
Received: 10 March 2017 / Revised: 30 May 2017 / Accepted: 30 May 2017 / Published: 6 June 2017
Cited by 1 | PDF Full-text (4669 KB) | HTML Full-text | XML Full-text | Supplementary Files
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
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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. Full article
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Review

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Open AccessReview A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis
Proteomes 2017, 5(2), 11; doi:10.3390/proteomes5020011
Received: 26 December 2016 / Revised: 4 April 2017 / Accepted: 4 April 2017 / Published: 7 April 2017
Cited by 2 | PDF Full-text (771 KB) | HTML Full-text | XML Full-text
Abstract
Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O′Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single
[...] Read more.
Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O′Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single ‘spots’ in a polyacrylamide gel, allowing the quantitation of changes in a proteoform′s abundance to ascertain changes in an organism′s phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the ‘Top-Down’. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O’Farrell’s paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism′s proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer. Full article
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