Relative Quantification of Proteins in Human Cerebrospinal Fluids by MS/MS Using 6-Plex Isobaric Tags
| Autor: | Virginie Licker, Jean-Charles Sanchez, Denis F. Hochstrasser, Loïc Dayon, Natacha Turck, Alexandre Hainard, Pierre Burkhard, Karsten Kuhn |
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| Rok vydání: | 2008 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Swine Trypsin/analysis Peptide Lactoglobulins S100 Calcium Binding Protein beta Subunit Nerve Growth Factors/cerebrospinal fluid Brain Injuries/cerebrospinal fluid Spectrometry Mass Electrospray Ionization/methods Tandem mass tag Mass spectrometry Tandem Mass Spectrometry/methods Analytical Chemistry Glial Fibrillary Acidic Protein/cerebrospinal fluid Cerebrospinal fluid Tandem Mass Spectrometry Creatine Kinase BB Form Glial Fibrillary Acidic Protein Milk/chemistry medicine Animals Humans Trypsin Horses Nerve Growth Factors ddc:576 Cerebrospinal Fluid/chemistry Cerebrospinal Fluid Cerebrospinal Fluid Proteins/analysis chemistry.chemical_classification Chromatography Myoglobin Chemistry S100 Proteins Lactoglobulins/analysis Cerebrospinal Fluid Proteins Creatine Kinase BB Form/cerebrospinal fluid S100 Proteins/cerebrospinal fluid Isobaric labeling Matrix-assisted laser desorption/ionization Milk Brain Injuries Isobaric process Cattle Myoglobin/analysis medicine.drug |
| Zdroj: | Analytical Chemistry, Vol. 80, No 8 (2008) pp. 2921-31 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/ac702422x |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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