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Identification of novel CSF biomarkers for neurodegeneration and their validation by a high-throughput multiplexed targeted proteomic assay.

Heywood WE, Galimberti D, Bliss E, Sirka E, Paterson RW, Magdalinou NK, Carecchio M, Reid E, Heslegrave A, Fenoglio C, Scarpini E, Schott JM, Fox NC, Hardy J, Bhatia K, Bahtia K, Heales S, Sebire NJ, Zetterberg H, Zetterburg H, Mills K - Mol Neurodegener (2015)

Bottom Line: Currently there are no effective treatments for many neurodegenerative diseases.Correlations with Alzheimer-associated amyloid β-42 levels, determined by ELISA, were observed for transthyretin, GM2 activator protein and IGF2 in the AD disease group (r(2) ≥ 0.39, p ≤ 0.012).This targeted proteomic platform can measure common markers of neurodegeneration that correlate with existing diagnostic makers as well as some that have potential to show changes between AD from LBD.

View Article: PubMed Central - PubMed

Affiliation: Centre for Translational Omics, University College London Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK. wendy.heywood@ucl.ac.uk.

ABSTRACT

Background: Currently there are no effective treatments for many neurodegenerative diseases. Reliable biomarkers for identifying and stratifying these diseases will be important in the development of future novel therapies. Lewy Body Dementia (LBD) is considered an under diagnosed form of dementia for which markers are needed to discriminate LBD from other forms of dementia such as Alzheimer's Disease (AD). This work describes a Label-Free proteomic profiling analysis of cerebral spinal fluid (CSF) from non-neurodegenerative controls and patients with LBD. Using this technology we identified several potential novel markers for LBD. These were then combined with other biomarkers from previously published studies, to create a 10 min multiplexed targeted and translational MRM-LC-MS/MS assay. This test was used to validate our new assay in a larger cohort of samples including controls and the other neurodegenerative conditions of Alzheimer's and Parkinson's disease (PD).

Results: Thirty eight proteins showed significantly (p < 0.05) altered expression in LBD CSF by proteomic profiling. The targeted MRM-LC-MS/MS assay revealed 4 proteins that were specific for the identification of AD from LBD: ectonucleotide pyrophosphatase/phosphodiesterase 2 (p < 0.0001), lysosome-associated membrane protein 1 (p < 0.0001), pro-orexin (p < 0.0017) and transthyretin (p < 0.0001). Nineteen proteins were elevated significantly in both AD and LBD versus the control group of which 4 proteins are novel (malate dehydrogenase 1, serum amyloid A4, GM2-activator protein, and prosaposin). Protein-DJ1 was only elevated significantly in the PD group and not in either LBD or AD samples. Correlations with Alzheimer-associated amyloid β-42 levels, determined by ELISA, were observed for transthyretin, GM2 activator protein and IGF2 in the AD disease group (r(2) ≥ 0.39, p ≤ 0.012). Cystatin C, ubiquitin and osteopontin showed a strong significant linear relationship (r(2) ≥ 0.4, p ≤ 0.03) with phosphorylated-tau levels in all groups, whilst malate dehydrogenase and apolipoprotein E demonstrated a linear relationship with phosphorylated-tau and total-tau levels in only AD and LBD disease groups.

Conclusions: Using proteomics we have identified several potential and novel markers of neurodegeneration and subsequently validated them using a rapid, multiplexed mass spectral test. This targeted proteomic platform can measure common markers of neurodegeneration that correlate with existing diagnostic makers as well as some that have potential to show changes between AD from LBD.

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Related in: MedlinePlus

Overlaid chromatogram of the marker peptides included in the multiplexed targeted proteomic assay. The assay was developed to quantitate 74 peptides in a 10 min LC run. Markers significant in the study are shown in the above overlaid chromatogram except for transferrin, serum amyloid A4 and apolipoprotein E which are not shown due to interference from other peaks. All markers are shown individually as endogenous and spiked in Additional file 8: Figure S2
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Fig2: Overlaid chromatogram of the marker peptides included in the multiplexed targeted proteomic assay. The assay was developed to quantitate 74 peptides in a 10 min LC run. Markers significant in the study are shown in the above overlaid chromatogram except for transferrin, serum amyloid A4 and apolipoprotein E which are not shown due to interference from other peaks. All markers are shown individually as endogenous and spiked in Additional file 8: Figure S2

Mentions: Table 1 summarises all the proteins that were found significantly altered compared to controls in this study. Based on fold-change (>1.5) and quality of the MS data, potential biomarkers identified in the discovery experiments were selected for validation by their development into a multiplexed 10 min, targeted proteomic triple quadrupole, peptide MRM-based assay. Chitinase-3-like protein 1 (YKL-40) and Apolipoprotein E were significant in the label free proteomics analyses but at a cut off of less than 1.5 fold. However as they have been described previously as neurodegeneration markers they were included in the assay to assess them with the other potential markers. Selected AD or PD specific markers described previously in the literature were also included [5–14] in order to compare with LBD. Using timed, or dynamic MRMs, in an UPLC-MS/MS assay, we created a method capable of analysing 74 peptides from 54 proteins using 100 μl of CSF. The full list of biomarkers put forward for validation is given in Additional file 3: Table S2. Out of these 54 potential biomarkers, only 27 resulted in being statistically significant and/or have potential for diagnostic use (Table 1). Fig. 2 includes an overlaid chromatogram of the UPLC-MS/MS chromatogram of the final successful targeted proteomic assay depicting the majority of the proteins/peptide biomarkers.Table 1


Identification of novel CSF biomarkers for neurodegeneration and their validation by a high-throughput multiplexed targeted proteomic assay.

Heywood WE, Galimberti D, Bliss E, Sirka E, Paterson RW, Magdalinou NK, Carecchio M, Reid E, Heslegrave A, Fenoglio C, Scarpini E, Schott JM, Fox NC, Hardy J, Bhatia K, Bahtia K, Heales S, Sebire NJ, Zetterberg H, Zetterburg H, Mills K - Mol Neurodegener (2015)

Overlaid chromatogram of the marker peptides included in the multiplexed targeted proteomic assay. The assay was developed to quantitate 74 peptides in a 10 min LC run. Markers significant in the study are shown in the above overlaid chromatogram except for transferrin, serum amyloid A4 and apolipoprotein E which are not shown due to interference from other peaks. All markers are shown individually as endogenous and spiked in Additional file 8: Figure S2
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666172&req=5

Fig2: Overlaid chromatogram of the marker peptides included in the multiplexed targeted proteomic assay. The assay was developed to quantitate 74 peptides in a 10 min LC run. Markers significant in the study are shown in the above overlaid chromatogram except for transferrin, serum amyloid A4 and apolipoprotein E which are not shown due to interference from other peaks. All markers are shown individually as endogenous and spiked in Additional file 8: Figure S2
Mentions: Table 1 summarises all the proteins that were found significantly altered compared to controls in this study. Based on fold-change (>1.5) and quality of the MS data, potential biomarkers identified in the discovery experiments were selected for validation by their development into a multiplexed 10 min, targeted proteomic triple quadrupole, peptide MRM-based assay. Chitinase-3-like protein 1 (YKL-40) and Apolipoprotein E were significant in the label free proteomics analyses but at a cut off of less than 1.5 fold. However as they have been described previously as neurodegeneration markers they were included in the assay to assess them with the other potential markers. Selected AD or PD specific markers described previously in the literature were also included [5–14] in order to compare with LBD. Using timed, or dynamic MRMs, in an UPLC-MS/MS assay, we created a method capable of analysing 74 peptides from 54 proteins using 100 μl of CSF. The full list of biomarkers put forward for validation is given in Additional file 3: Table S2. Out of these 54 potential biomarkers, only 27 resulted in being statistically significant and/or have potential for diagnostic use (Table 1). Fig. 2 includes an overlaid chromatogram of the UPLC-MS/MS chromatogram of the final successful targeted proteomic assay depicting the majority of the proteins/peptide biomarkers.Table 1

Bottom Line: Currently there are no effective treatments for many neurodegenerative diseases.Correlations with Alzheimer-associated amyloid β-42 levels, determined by ELISA, were observed for transthyretin, GM2 activator protein and IGF2 in the AD disease group (r(2) ≥ 0.39, p ≤ 0.012).This targeted proteomic platform can measure common markers of neurodegeneration that correlate with existing diagnostic makers as well as some that have potential to show changes between AD from LBD.

View Article: PubMed Central - PubMed

Affiliation: Centre for Translational Omics, University College London Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK. wendy.heywood@ucl.ac.uk.

ABSTRACT

Background: Currently there are no effective treatments for many neurodegenerative diseases. Reliable biomarkers for identifying and stratifying these diseases will be important in the development of future novel therapies. Lewy Body Dementia (LBD) is considered an under diagnosed form of dementia for which markers are needed to discriminate LBD from other forms of dementia such as Alzheimer's Disease (AD). This work describes a Label-Free proteomic profiling analysis of cerebral spinal fluid (CSF) from non-neurodegenerative controls and patients with LBD. Using this technology we identified several potential novel markers for LBD. These were then combined with other biomarkers from previously published studies, to create a 10 min multiplexed targeted and translational MRM-LC-MS/MS assay. This test was used to validate our new assay in a larger cohort of samples including controls and the other neurodegenerative conditions of Alzheimer's and Parkinson's disease (PD).

Results: Thirty eight proteins showed significantly (p < 0.05) altered expression in LBD CSF by proteomic profiling. The targeted MRM-LC-MS/MS assay revealed 4 proteins that were specific for the identification of AD from LBD: ectonucleotide pyrophosphatase/phosphodiesterase 2 (p < 0.0001), lysosome-associated membrane protein 1 (p < 0.0001), pro-orexin (p < 0.0017) and transthyretin (p < 0.0001). Nineteen proteins were elevated significantly in both AD and LBD versus the control group of which 4 proteins are novel (malate dehydrogenase 1, serum amyloid A4, GM2-activator protein, and prosaposin). Protein-DJ1 was only elevated significantly in the PD group and not in either LBD or AD samples. Correlations with Alzheimer-associated amyloid β-42 levels, determined by ELISA, were observed for transthyretin, GM2 activator protein and IGF2 in the AD disease group (r(2) ≥ 0.39, p ≤ 0.012). Cystatin C, ubiquitin and osteopontin showed a strong significant linear relationship (r(2) ≥ 0.4, p ≤ 0.03) with phosphorylated-tau levels in all groups, whilst malate dehydrogenase and apolipoprotein E demonstrated a linear relationship with phosphorylated-tau and total-tau levels in only AD and LBD disease groups.

Conclusions: Using proteomics we have identified several potential and novel markers of neurodegeneration and subsequently validated them using a rapid, multiplexed mass spectral test. This targeted proteomic platform can measure common markers of neurodegeneration that correlate with existing diagnostic makers as well as some that have potential to show changes between AD from LBD.

Show MeSH
Related in: MedlinePlus