Comparison of Hydrophobic, Lipophilic and Immunodepletion Pre- Fractionation Methods for Label-Free LC-MS/MS Identification of Biomarkers in Human Cerebrospinal Fluid - Université de Montpellier Accéder directement au contenu
Article Dans Une Revue Journal of Proteomics and Bioinformatics Année : 2015

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

Résumé

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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hal-03108550 , version 1 (13-01-2021)

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Paternité - Pas d'utilisation commerciale

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Sylvain Lehmann, Jérôme Vialaret, Martial Séveno, Laurent Tiers, Audrey Gabelle, et al.. Comparison of Hydrophobic, Lipophilic and Immunodepletion Pre- Fractionation Methods for Label-Free LC-MS/MS Identification of Biomarkers in Human Cerebrospinal Fluid. Journal of Proteomics and Bioinformatics, 2015, s5, ⟨10.4172/JPB.S5-003⟩. ⟨hal-03108550⟩
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