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Affinity depletion versus relative protein enrichment: a side-by-side comparison of two major strategies for increasing human cerebrospinal fluid proteome coverage.

ABSTRACT: Cerebrospinal fluid (CSF) is in direct contact with the central nervous system. This makes human CSF an attractive source of potential biomarkers for neurologic diseases. Similarly to blood plasma, proteomic analysis of CSF is complicated by a high dynamic range of individual protein concentrations and by the presence of several highly abundant proteins. To deal with the abundant human CSF proteins, methods developed for blood plasma/serum are routinely used. Multiple affinity removal systems and protein enrichment of less abundant proteins using a combinatorial peptide ligand library are among the most frequent approaches. However, their relative impact on CSF proteome coverage has never been evaluated side-by-side in a single study. Therefore, we explored the effect of CSF depletion using MARS 14 cartridge and ProteoMiner ligand library on the number of CSF proteins identified in subsequent LC-MS/MS analysis. LC-MS/MS analysis of crude (non-treated) CSF provided roughly 500 identified proteins. Depletion of CSF by MARS 14 cartridge increased the number of identifications to nearly 800, while treatment of CSF using ProteoMiner enabled identification of 600 proteins. To explore the potential losses of CSF proteins during the depletion process, we also analyzed the "waste" fractions generated by both methods, i.e., proteins retained by the MARS 14 cartridge, and the molecules present in the flow-through fraction from ProteoMiner. More than 250 proteins were bound to MARS 14 cartridge, 100 of those were not identified in the corresponding depleted CSF. Similarly, analysis of the waste fraction in ProteoMiner workflow provided almost 70 unique proteins not found in the CSF depleted by the ligand library. Both depletion strategies significantly increased the number of identified CSF proteins compared to crude CSF. However, MARS 14 depletion provided a markedly higher number of identified proteins (773) compared to ProteoMiner (611). Further, we showed that CSF proteins are lost due to co-depletion (MARS 14) or exclusion (ProteoMiner) during the depletion process. This suggests that the routinely discarded "waste" fractions contain proteins of potential interest and should be included in CSF biomarker studies.

SUBMITTER: Jankovska E

PROVIDER: S-EPMC6390343 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Affinity depletion versus relative protein enrichment: a side-by-side comparison of two major strategies for increasing human cerebrospinal fluid proteome coverage.

Jankovska Eliska E Svitek Marek M Holada Karel K Petrak Jiri J

Clinical proteomics 20190226

Cerebrospinal fluid (CSF) is in direct contact with the central nervous system. This makes human CSF an attractive source of potential biomarkers for neurologic diseases. Similarly to blood plasma, proteomic analysis of CSF is complicated by a high dynamic range of individual protein concentrations and by the presence of several highly abundant proteins. To deal with the abundant human CSF proteins, methods developed for blood plasma/serum are routinely used. Multiple affinity removal systems an ...[more]

PMID: 30890900

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Project description:BackgroundAltered proteome profiles have been reported in both postmortem brain tissues and body fluids of subjects with Alzheimer disease (AD), but their broad relationships with AD pathology, amyloid pathology, and tau-related neurodegeneration have not yet been fully explored. Using a robust automated MS-based proteomic biomarker discovery workflow, we measured cerebrospinal fluid (CSF) proteomes to explore their association with well-established markers of core AD pathology.MethodsCross-sectional analysis was performed on CSF collected from 120 older community-dwelling adults with normal (n?=?48) or impaired cognition (n?=?72). LC-MS quantified hundreds of proteins in the CSF. CSF concentrations of ?-amyloid 1-42 (A?1-42), tau, and tau phosphorylated at threonine 181 (P-tau181) were determined with immunoassays. First, we explored proteins relevant to biomarker-defined AD. Then, correlation analysis of CSF proteins with CSF markers of amyloid pathology, neuronal injury, and tau hyperphosphorylation (i.e., A?1-42, tau, P-tau181) was performed using Pearson's correlation coefficient and Bonferroni correction for multiple comparisons.ResultsWe quantified 790 proteins in CSF samples with MS. Four CSF proteins showed an association with CSF A?1-42 levels (p value ??0.05 with correlation coefficient (R)???0.38). We identified 50 additional CSF proteins associated with CSF tau and 46 proteins associated with CSF P-tau181 (p value ??0.05 with R???0.37). The majority of those proteins that showed such associations were brain-enriched proteins. Gene Ontology annotation revealed an enrichment for synaptic proteins and proteins originating from reelin-producing cells and the myelin sheath.ConclusionsWe used an MS-based proteomic workflow to profile the CSF proteome in relation to cerebral AD pathology. We report strong evidence of previously reported CSF proteins and several novel CSF proteins specifically associated with amyloid pathology or neuronal injury and tau hyperphosphorylation.

Dataset Information

Affinity depletion versus relative protein enrichment: a side-by-side comparison of two major strategies for increasing human cerebrospinal fluid proteome coverage.

Publications

Affinity depletion versus relative protein enrichment: a side-by-side comparison of two major strategies for increasing human cerebrospinal fluid proteome coverage.

Similar Datasets

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