alexa Comparison of Hydrophobic, Lipophilic and Immunodepletion Pre- Fractionation Methods for Label-Free LC-MS/MS Identification of Biomarkers in Human Cerebrospinal Fluid
ISSN: 0974-276X

Journal of Proteomics & Bioinformatics
Open Access

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Research Article

Comparison of Hydrophobic, Lipophilic and Immunodepletion Pre- Fractionation Methods for Label-Free LC-MS/MS Identification of Biomarkers in Human Cerebrospinal Fluid

Sylvain Lehmann1*#, Jérôme Vialaret1#, Martial Seveno2, Laurent Tiers1, Audrey Gabelle1,3 and Christophe Hirtz1*

1CHU Montpellier, Institute of Research in Biotherapy, Hôpital Saint Eloi, Laboratory of Biochemistry and Clinical Proteomics CCBHM, Montpellier, INSERM U1040-UM1, Montpellier, F-34000 France

2Platform Functional Proteomics, IGF, CNRS UMR 5203, INSERM U661, Université Montpellier I and II, Montpellier, France

3Memory Resources Research Centre, CHU Montpellier, Gui de Chauliac Hospital, Montpellier. Montpellier I University, Montpellier, F-34000 France

#These authors contributed equally to the manuscript

*Corresponding Author:
Sylvain Lehmann
CHU Montpellier, Institute of Research in Biotherapy
Hôpital Saint Eloi, Laboratory of Biochemistry and Clinical Proteomics CCBHM
Montpellier, INSERM U1040-UM1, Montpellier, F-34000 France
Tel: +33 603422349
E-mail: [email protected]

Christophe Hirtz
CHU Montpellier, Institute of Research in Biotherapy
Hôpital Saint Eloi, Laboratory of Biochemistry and Clinical Proteomics CCBHM
Montpellier, INSERM U1040-UM1, Montpellier, F-34000 France
Tel: +33 603422349
E-mail: [email protected]

Received date: March 09, 2014; Accepted date: April 17, 2014; Published date: April 21, 2014

Citation: Lehmann S, Vialaret J, Seveno M, Tiers L, Gabelle A, et al. (2014) Comparison of Hydrophobic, Lipophilic and Immunodepletion Pre-Fractionation Methods for Label-Free LC-MS/MS Identification of Biomarkers in Human Cerebrospinal Fluid. J Proteomics Bioinform S5:003. doi: 10.4172/jpb.S5-003

Copyright: © 2014 Lehmann S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Background: Proteomics analysis of human cerebrospinal fluid (CSF) is a major tool for identifying novel biomarkers for neurological diseases. However, the complexity and wide dynamic range of CSF represent a major challenge for detecting specific low-abundance biomarkers. One way to overcome this problem is to rely on different pre-fractionation techniques. However, the most relevant technique remains to be determined.

Methods: This study compared three different well-known pre-fractionation methods: immuno-depletion of major proteins (Seppro® IgY14), hydrophobic solid phase extraction (Oasis® HLB), and lipophilic sorbent concentration (Liposorb™). Unfractionated and pre-fractionated CSF was digested with trypsin and analyzed by RP-LC-MS/ MS with an OrbitrapTM mass spectrometer. We documented the number of peptides detected and sets of proteins identified. Experiments were repeated to minimize pre-analytical and analytical variability.

Results: Compared to unfractionated CSF, the OASIS® HLB fractionated CSF method showed a significant 28% increase in the total number of proteins identified, while the Liposorb™ capture resulted in a significant 46% decrease. Interestingly, results based on the number of peptides detected were different. We also evaluated the capacity of these pre-fractionation methods to detect different proteins in terms of their molecular weight, isoelectrophoretic point (IEP) or nature. Each of these pre-fractionation methods identified a specific subset of proteins, when compared to unfractionated CSF, and/or other methods. This was particularly obvious for the lipophilic sorbent, which allowed the detection of many lipoproteins.

Conclusion: Direct analysis of digested CSF led to the identification of several proteins despite matrix complexity. As expected, single pre-fractionation methods that can be included in simple and cost-effective workflows, yielded significant differences in terms of number, or range of proteins identified. This suggests that a single pre-fractionation method cannot cover the full range of protein species present in a complex sample.

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