Spectral counting assessment of protein dynamic range in cerebrospinal fluid following depletion with plasma-designed immunoaffinity columns

Clinical Proteomics

Núria Omeñaca⁴  Eliandre de Oliveira¹  Joan Guinovart²  Alex Campos¹  Claudio Diema⁴  Jacques Borg³  Marta Vilaseca⁴ 

[1] Proteomics Platform, Barcelona, SpainProteomics Platform, Barcelona, SpainProteomics Platform, Barcelona, Spain;Institute for Research in Biomedicine, Barcelona, SpainInstitute for Research in Biomedicine, Barcelona, SpainInstitute for Research in Biomedicine, Barcelona, Spain;Laboratoire de Neurobiochimie, Université Jean Monnet, Saint-Etienne, FranceLaboratoire de Neurobiochimie, Université Jean Monnet, Saint-Etienne, FranceLaboratoire de Neurobiochimie, Université Jean Monnet, Saint-Etienne, France;Mass Spectrometry Core Facility, Institute for Research in Biomedicine, Barcelona, SpainMass Spectrometry Core Facility, Institute for Research in Biomedicine, Barcelona, SpainMass Spectrometry Core Facility, Institute for Research in Biomedicine, Barcelona, Spain

关键词: CSF;    APEX;    Biomarkers;    depletion column;    enrichment;    low-abundance proteins;   

DOI  :  10.1186/1559-0275-8-6

来源: Humana Press Inc

【 摘 要 】

Abstract

Background

In cerebrospinal fluid (CSF), which is a rich source of biomarkers for neurological diseases, identification of biomarkers requires methods that allow reproducible detection of low abundance proteins. It is therefore crucial to decrease dynamic range and improve assessment of protein abundance.

Results

We applied LC-MS/MS to compare the performance of two CSF enrichment techniques that immunodeplete either albumin alone (IgYHSA) or 14 high-abundance proteins (IgY14). In order to estimate dynamic range of proteins identified, we measured protein abundance with APEX spectral counting method. Both immunodepletion methods improved the number of low-abundance proteins detected (3-fold for IgYHSA, 4-fold for IgY14). The 10 most abundant proteins following immunodepletion accounted for 41% (IgY14) and 46% (IgYHSA) of CSF protein content, whereas they accounted for 64% in non-depleted samples, thus demonstrating significant enrichment of low-abundance proteins. Defined proteomics experiment metrics showed overall good reproducibility of the two immunodepletion methods and MS analysis. Moreover, offline peptide fractionation in IgYHSA sample allowed a 4-fold increase of proteins identified (520 vs. 131 without fractionation), without hindering reproducibility.

Conclusions

The novelty of this study was to show the advantages and drawbacks of these methods side-to-side. Taking into account the improved detection and potential loss of non-target proteins following extensive immunodepletion, it is concluded that both depletion methods combined with spectral counting may be of interest before further fractionation, when searching for CSF biomarkers. According to the reliable identification and quantitation obtained with APEX algorithm, it may be considered as a cheap and quick alternative to study sample proteomic content.

【 授权许可】

Unknown