Abstract

BackgroundTo date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries.Methodology/Principal FindingsHere we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration.Conclusions/SignificanceOur results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol.

Highlights

  • The human blood is a rich source for biomarker discovery

  • Characterization of the human plasma proteome is a very difficult task: the top ten most abundant plasma proteins account for approximately 90% of the total protein content, while other proteins are present in a very wide dynamic range, spanning more than 10 orders of magnitude in terms of concentration [3]

  • The aim of this study was to determine which method between high abundance proteins (HAPs) depletion and low abundance proteins (LAPs) enrichment provides the best overall results in terms of number of identified proteins, protein coverage and enhanced sensitivity limit

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Summary

Introduction

The human blood is a rich source for biomarker discovery. Plasma is usually preferred over serum for the lower ex vivo protein degradation [1,2].A comprehensive, systematic characterization of plasma proteome in healthy and diseased states could greatly facilitate the detection of biomarkers for early disease diagnosis, prognosis and therapeutic monitoring. Characterization of the human plasma proteome is a very difficult task: the top ten most abundant plasma proteins account for approximately 90% of the total protein content, while other proteins are present in a very wide dynamic range, spanning more than 10 orders of magnitude in terms of concentration [3]. This last feature, in particular, makes the plasma proteome the most complex human-derived proteome. The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries

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