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Title

Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags

Authors
Kuhn, Karsten
Published in Analytical Chemistry. 2008, vol. 80, no. 8, p. 2921-31
Abstract A new 6-plex isobaric mass tagging technology is presented, and proof of principle studies are carried out using standard protein mixtures and human cerebrospinal fluid (CSF) samples. The Tandem Mass Tags (TMT) comprise a set of structurally identical tags which label peptides on free amino-terminus and epsilon-amino functions of lysine residues. During MS/MS fragmentation, quantification information is obtained through the losses of the reporter ions. After evaluation of the relative quantification with the 6-plex version of the TMT on a model protein mixture at various concentrations, the quantification of proteins in CSF samples was performed using shotgun methods. Human postmortem (PM) CSF was taken as a model of massive brain injury and comparison was carried out with antemortem (AM) CSF. After immunoaffinity depletion, triplicates of AM and PM CSF pooled samples were reduced, alkylated, digested by trypsin, and labeled, respectively, with the six isobaric variants of the TMT (with reporter ions from m/z = 126.1 to 131.1 Th). The samples were pooled and fractionated by SCX chromatography. After RP-LC separation, peptides were identified and quantified by MS/MS analysis with MALDI TOF/TOF and ESI-Q-TOF. The concentration of 78 identified proteins was shown to be clearly increased in PM CSF samples compared to AM. Some of these proteins, like GFAP, protein S100B, and PARK7, have been previously described as brain damage biomarkers, supporting the PM CSF as a valid model of brain insult. ELISA for these proteins confirmed their elevated concentration in PM CSF. This work demonstrates the validity and robustness of the tandem mass tag (TMT) approach for quantitative MS-based proteomics.
Keywords AnimalsBrain Injuries/cerebrospinal fluidCattleCerebrospinal Fluid/chemistryCerebrospinal Fluid Proteins/analysisCreatine Kinase, BB Form/cerebrospinal fluidGlial Fibrillary Acidic Protein/cerebrospinal fluidHorsesHumansLactoglobulins/analysisMilk/chemistryMyoglobin/analysisNerve Growth Factors/cerebrospinal fluidS100 Proteins/cerebrospinal fluidSpectrometry, Mass, Electrospray Ionization/methodsSwineTandem Mass Spectrometry/methodsTrypsin/analysis
Identifiers
PMID: 18312001
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Research groups Groupe Pierre Burkhard (neurologie) (154)
La protéomique au service de la clinique (270)
Groupe de Protéomique biomédicale (635)
Citation
(ISO format)
DAYON, Loic et al. Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags. In: Analytical Chemistry, 2008, vol. 80, n° 8, p. 2921-31. https://archive-ouverte.unige.ch/unige:901

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Deposited on : 2009-02-24

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