A Routine 'Top-Down' Approach to Analysis of the Human Serum Proteome.

Arlene D’Silva,Jon Hyett,Jens Coorssen

doi:10.3390/proteomes5020013

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.

Highlights

Detailed analyses of the serum proteome are important as they provide a source of diagnostic or prognostic biomarkers as well as insight into the mechanisms underlying disease development and progression [1,2]
When selecting a prefractionation method to assist in sample preparation, it is imperative to assess the potential for loss of low abundance proteins; the possibility of carrier protein interactions with critical low molecular weight biomarkers is likely a serious issue hampering further advances
We will be applying the approach defined here to further characterize and quantify protein species in a larger cohort of subjects to provide a better understanding of the maternal serum proteome

Summary

Introduction

Detailed analyses of the serum proteome are important as they provide a source of diagnostic or prognostic biomarkers as well as insight into the mechanisms underlying disease development and progression [1,2]. Proteomes 2017, 5, 13 proteoforms in a single analytical run, while enabling multiple parallel analyses As the only such routine top-down analytical protocol, it is the only available approach that enables quantitative profiling of large sets of complex protein mixtures; that is, as part of the routine analytical protocol, this approach resolves protein isoforms, splice variants, and the vast range of post-translationally modified protein species that define biological functionality. High-abundance proteins tend to mask those of lower abundance and have typically been removed to allow better resolution of other species [8] Removal of this fraction risks removal of non-targeted proteins that may impact on our understanding of the mechanisms underlying disease as well as on discovery and quantification of novel biomarkers [9]. Removal of the most abundant proteins merely exposes a second cohort that is highly abundant in comparison to other species—so this intervention fails to resolve the fundamental problem of dynamic range and largely obviates the objective of quantitative analysis [5]

Methods

Discussion

Conclusion