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

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	CSFColiCalibrationCurve_InColi.xml	Xml
	CSFColiCalibrationCurve_InHuman.xml	Xml
	CSF_Evaluation_Depleted_NonDepleted.xml	Xml
	CSF_Replicate1_01.raw	Raw
	CSF_Replicate1_02.raw	Raw

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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: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).