Characterization and stability of transthyretin isoforms in cerebrospinal fluid examined by immunoprecipitation and high-resolution mass spectrometry of intact protein
| Autor: | Keld Poulsen, Anja Hviid Simonsen, Julia T. Tanassi, Niels H. H. Heegaard, Justyna M.C. Bahl |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
Gene isoform
endocrine system Immunoprecipitation Diagnostic Techniques Neurological medicine.disease_cause General Biochemistry Genetics and Molecular Biology Antibodies Mass Spectrometry Specimen Handling Cerebrospinal fluid Alzheimer Disease medicine Humans Prealbumin Protein Isoforms Molecular Biology biology Chemistry Protein Stability Amyloidosis Temperature nutritional and metabolic diseases medicine.disease Transthyretin Freeze Drying Biochemistry Case-Control Studies biology.protein Alzheimer's disease Cognition Disorders Oxidation-Reduction Oxidative stress Cysteine |
| Zdroj: | Methods (San Diego, Calif.). 56(2) |
| ISSN: | 1095-9130 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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