title: Characterization and stability of transthyretin isoforms in cerebrospinal fluid examined by immunoprecipitation and high-resolution mass spectrometry of intact protein.
abstract: Post-translational modifications (PTMs) contribute significantly to the complexity of proteins. PTMs may vary in certain patterns according to diseases and microenviroments making them potential markers for pathological processes. Human transthyretin (TTR) is a transporter of thyroxine and retinol in blood and cerebrospinal fluid (CSF). A single free cysteine thiol group in TTR possesses the ability to form mixed disulfides potentially related to diseases such as TTR amyloidosis and Alzheimer's disease (AD). Additionally, TTR-Cys10 S-thiolations might mirror the oxidative stress and redox balance of CSF. Here we describe a quick and gentle method for immunoprecipitating (IP) TTR from CSF with minimal introduction of sample-handling artifacts. A high-resolution mass spectrometer (LTQ-Orbitrap XL) was used in a simple setup with direct infusion that generates data suitable for confident assignment of TTR isoforms and validation of the protocol. Moreover, we demonstrate how simple storage of CSF at 4°C induces major oxidative modifications of TTR. Using the optimized method, we show data from a limited number of mild cognitive impairment (MCI) and AD patients. The protocol controls and minimizes the introduction of sample-handling artifacts during purification of TTR isoforms for high-resolution MS analysis.
authors.affiliation: Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen, Denmark. kpl@ssi.dk
authors.forename: Keld
authors.initials: K
authors.lastname: Poulsen
authors.forename: Justyna M C
authors.initials: JM
authors.lastname: Bahl
authors.forename: Julia T
authors.initials: JT
authors.lastname: Tanassi
authors.forename: Anja H
authors.initials: AH
authors.lastname: Simonsen
authors.forename: Niels H H
authors.initials: NH
authors.lastname: Heegaard
mesh_terms: D000544:Alzheimer Disease / Q000134:cerebrospinal fluid; D000906:Antibodies / Q000737:chemistry; D016022:Case-Control Studies; D003072:Cognition Disorders / Q000134:cerebrospinal fluid; D003943:Diagnostic Techniques, Neurological; D005612:Freeze Drying; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D010084:Oxidation-Reduction; D011228:Prealbumin / Q000134:cerebrospinal fluid* / Q000737:chemistry / Q000302:isolation & purification; D020033:Protein Isoforms / Q000134:cerebrospinal fluid / Q000737:chemistry / Q000302:isolation & purification; D055550:Protein Stability; D013048:Specimen Handling / Q000379:methods; D013696:Temperature
chemical_list: D000906:Antibodies; D011228:Prealbumin; D020033:Protein Isoforms