Dataset Information

Proteomics of Cerebrospinal Fluid: Throughput and Robustness Using a Scalable Automated Analysis Pipeline for Biomarker Discovery

ABSTRACT: Cerebrospinal fluid (CSF) is a body fluid of high clinical relevance and an important source of potential biomarkers for brain-associated damages such as traumatic brain injury, stroke, and for brain diseases like Alzheimer’s and Parkinson's . Herein, we have implemented, evaluated and validated a scalable automated proteomic pipeline (ASAP2) for the sample preparation and proteomic analysis of CSF, enabling increased throughput and robustness for biomarker discovery. Human CSF samples were depleted from abundant proteins, and submitted to automated reduction, alkylation, protein digestion, tandem mass tag (TMT) 6-plex labeling, pooling, and sample clean-up in a 96-well plate format before reversed-phase liquid chromatography tandem mass spectrometry (RP-LC MS/MS). First, we showed the impact on the CSF proteome coverage of applying the depletion of abundant proteins, which is usually performed on blood plasma or serum samples. Using ASAP2 to analyze 96 identical CSF samples, we determined the analytical figures of merit of our shotgun proteomic approach regarding proteome coverage consistency (i.e., 387 proteins), quantitative accuracy, and individual protein variability. We demonstrated that, as for human plasma samples, ASAP2 is efficient in analyzing large numbers of human CSF samples and is a valuable tool for biomarker discovery.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cerebrospinal Fluid

SUBMITTER: Loïc Dayon

LAB HEAD: Loïc Dayon

PROVIDER: PXD003024 | Pride | 2015-12-09

REPOSITORIES: Pride

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Publications

Proteomics of Cerebrospinal Fluid: Throughput and Robustness Using a Scalable Automated Analysis Pipeline for Biomarker Discovery.

Núñez Galindo Antonio A Kussmann Martin M Dayon Loïc L

Analytical chemistry 20151016 21

Cerebrospinal fluid (CSF) is a body fluid of high clinical relevance and an important source of potential biomarkers for brain-associated damages, such as traumatic brain injury and stroke, and for brain diseases, such as Alzheimer's and Parkinson's. Herein, we have implemented, evaluated, and validated a scalable automated proteomic pipeline (ASAP(2)) for the sample preparation and proteomic analysis of CSF, enabling increased throughput and robustness for biomarker discovery. Human CSF samples ...[more]

PMID: 26452177

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Project description:Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurodegenerative disease that affects motor, urinary, intestinal, and sensory functions. Typically, HAM/TSP is slowly progressive, but it may vary from limited motor disability after decades (very slow progression) to loss of motor function in a few years from disease onset (rapid). In this study, we aimed to identify prognostic biomarkers for HAM/TSP to support patient management. Thus, proteomic analysis of the cerebrospinal fluid (CSF) was performed with samples from HTLV-1 asymptomatic carriers (AC) (n=13) and HAM/TSP patients (n=21) with rapid, typical, and very slow progression using quantitative label-free liquid chromatography/tandem mass spectrometry. Enrichment analyses were also carried out to identify key biological processes associated with distinct neurological conditions in HTLV-1 infection. Candidate biomarkers were validated by ELISA in paired CSF and serum samples, and samples from HTLV-1-seronegative individuals (n=9) were used as controls. CSF analysis identified 602 proteins. Leukocyte/cell activation, immune response processes and neurodegeneration pathways were enriched in rapid progressors. Conversely, HTLV-1 AC and HAM/TSP patients with typical and very slow progression had enriched processes for nervous system development. Differential expression analysis showed that soluble vascular cell adhesion molecule 1 (sVCAM-1), chitotriosidase 1 (CHIT1), and cathepsin C (CTSC) were upregulated in HAM/TSP. However, only CHIT1 was significantly elevated after validation, particularly in HAM/TSP rapid progressors. In contrast, none of these biomarkers were altered in serum. Additionally, CSF CHIT1 levels in HAM/TSP patients positively correlated with the speed of HAM/TSP progression, defined as points in the IPEC-2 HAM/TSP disability scale per year of disease, and with CSF levels of phosphorylated neurofilament heavy chain, neopterin, CXCL5, CXCL10, and CXCL11. In conclusion, higher CSF levels of CHIT1 were associated with HAM/TSP rapid progression and correlated with other biomarkers of neuroinflammation and neurodegeneration. Therefore, we propose CHIT1 as a surrogate CSF biomarker to identify HAM/TSP patients with a worse prognosis.

Project description:Being inspired by metabolomic data processing, we have developed a bioinformatic pipeline that optimizes the processing of mass spectral data obtained from isobaric Tandem Mass Tag (TMT) experiments. Our method focuses on the tandem mass spectral level by first quantifying and then identifying (QtI), while preserving unidentified spectra for further investigations. The raw datasets were previously generated [1, 2]. Two-proteome model experiments were considered where identical pools of human CSF or plasma samples were mixed with E. coli samples at different concentrations. E. coli protein extract was spiked in 400 µL of CSF at amounts of 0, 2, 3, 5, 6.25, and 7.5 µg. Such sets of 6 spiked CSF samples were prepared in triplicate for comparison using sixplex isobaric tagging and analyzed in triplicates on two independent but identical LC MS/MS, for a total of 18 raw files [1]; this experiment is called “CSF-E.coli”. E. coli protein extract was spiked at 0, 2.5, 5, 6.25, 12.5, and 25 µg in 30 µL human plasma. Such sets of 6 spiked plasma samples were prepared in quadruplicate for comparison using sixplex isobaric tagging and analyzed in triplicates on one LC MS/MS, for a total of 12 raw files [2]; this experiment is referred as “Plasma-E.coli”.The so-called “96samples-CSF” experiment consists of 16 replicate TMT sixplex experiments measuring identical CSF samples from the pool described above [2], analyzed in triplicates on one LCMS/MS for a total of 48 raw files. The so-called “96samples-plasma” experiment consists of 16 replicate TMT sixplex experiments measuring identical plasma samples from the pool described before, analyzed in triplicates on one LC MS/MS for a total of 48 raw files [1]. References: [1] Dayon, L., Núñez Galindo, A., Corthésy, J., Cominetti, O. & Kussmann, M. Comprehensive and scalable highly automated MS-based proteomic workflow for clinical biomarker discovery in human plasma. J. Proteome Res. 13, 3837-3845 (2014). [2] Núñez Galindo, A., Kussmann, M. & Dayon, L. Proteomics of Cerebrospinal Fluid: Throughput and Robustness Using a Scalable Automated Analysis Pipeline for Biomarker Discovery. Anal. Chem. 87, 10755-10761 (2015).

Dataset Information

Proteomics of Cerebrospinal Fluid: Throughput and Robustness Using a Scalable Automated Analysis Pipeline for Biomarker Discovery

Publications

Proteomics of Cerebrospinal Fluid: Throughput and Robustness Using a Scalable Automated Analysis Pipeline for Biomarker Discovery.

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