title: Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags.
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.
authors.affiliation: Biomedical Proteomics Group, Department of Structural Biology and Bioinformatics, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
authors.forename: Loïc
authors.initials: L
authors.lastname: Dayon
authors.forename: Alexandre
authors.initials: A
authors.lastname: Hainard
authors.forename: Virginie
authors.initials: V
authors.lastname: Licker
authors.forename: Natacha
authors.initials: N
authors.lastname: Turck
authors.forename: Karsten
authors.initials: K
authors.lastname: Kuhn
authors.forename: Denis F
authors.initials: DF
authors.lastname: Hochstrasser
authors.forename: Pierre R
authors.initials: PR
authors.lastname: Burkhard
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Sanchez
mesh_terms: D000818:Animals; D001930:Brain Injuries / Q000134:cerebrospinal fluid; D002417:Cattle; D002555:Cerebrospinal Fluid / Q000737:chemistry; D002556:Cerebrospinal Fluid Proteins / Q000032:analysis*; D052276:Creatine Kinase, BB Form / Q000134:cerebrospinal fluid; D005904:Glial Fibrillary Acidic Protein / Q000134:cerebrospinal fluid; D006736:Horses; D006801:Humans; D007782:Lactoglobulins / Q000032:analysis; D008892:Milk / Q000737:chemistry; D009211:Myoglobin / Q000032:analysis; D009414:Nerve Growth Factors / Q000134:cerebrospinal fluid; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000134:cerebrospinal fluid; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000032:analysis
chemical_list: D002556:Cerebrospinal Fluid Proteins; D005904:Glial Fibrillary Acidic Protein; D007782:Lactoglobulins; D009211:Myoglobin; D009414:Nerve Growth Factors; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579160:S100B protein, human; D052276:Creatine Kinase, BB Form; D014357:Trypsin