Automated sample preparation platform for mass spectrometry-based plasma proteomics and biomarker discovery.

Vilém Guryča,Daniel Roeder,Paul Cutler,Jens Lamerz,Axel Ducret,Paolo Piraino,Hanno Langen

doi:10.3390/biology3010205

Abstract

The identification of novel biomarkers from human plasma remains a critical need in order to develop and monitor drug therapies for nearly all disease areas. The discovery of novel plasma biomarkers is, however, significantly hampered by the complexity and dynamic range of proteins within plasma, as well as the inherent variability in composition from patient to patient. In addition, it is widely accepted that most soluble plasma biomarkers for diseases such as cancer will be represented by tissue leakage products, circulating in plasma at low levels. It is therefore necessary to find approaches with the prerequisite level of sensitivity in such a complex biological matrix. Strategies for fractionating the plasma proteome have been suggested, but improvements in sensitivity are often negated by the resultant process variability. Here we describe an approach using multidimensional chromatography and on-line protein derivatization, which allows for higher sensitivity, whilst minimizing the process variability. In order to evaluate this automated process fully, we demonstrate three levels of processing and compare sensitivity, throughput and reproducibility. We demonstrate that high sensitivity analysis of the human plasma proteome is possible down to the low ng/mL or even high pg/mL level with a high degree of technical reproducibility.

Highlights

Due to their sensitivity and multiplexing capabilities, mass spectrometry (MS) based proteomics approaches have emerged as one of the indispensable tools for biomarker discovery in clinical research [1]
We have developed and evaluated a generic sample preparation platform, targeting critical biological samples, which works at a micro-scale and is sufficiently sensitive to detect protein levels significantly below 1 μg/mL
In order to establish a new methodology for biomarker discovery in human plasma, we have created a novel semi-automated multidimensional chromatography process, combining most of the manual steps used in common proteomics protocols

Summary

Introduction

Due to their sensitivity and multiplexing capabilities, mass spectrometry (MS) based proteomics approaches have emerged as one of the indispensable tools for biomarker discovery in clinical research [1]. Plasma proteomics by mass spectrometry remains one of the few available methods to systematically characterize molecular alterations at the protein level in an unbiased way [2]. It has long been recognized that sample preparation and fractionation is key to successful profiling of the plasma proteome. The delivery of the peptides to the mass spectrometer is often coupled to a nanoflow chromatography system. Despite the increased sensitivity concomitant with nanoflow chromatography the ability to detect low abundant proteins in plasma is still challenging. To increase the sensitivity of the analysis further, various fractionation techniques can be employed, either at the peptide or the protein level. Some traditional protein fractionation methods, such as gel electrophoresis have been employed but have finite reproducibility, caused, in particular, by manual handling of samples [3]

Methods

Results

Conclusion