Robust two-dimensional separation of intact proteins for bottom-up tandem mass spectrometry of the human CSF proteome
| Autor: | Carol Anderson, Muznabanu Bachani, Robert J. Cotter, Avindra Nath, Adriana Bora |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Chromatography
Reverse-Phase Spectrometry Mass Electrospray Ionization Chromatography Proteome Chemistry Electrospray ionization Cerebrospinal Fluid Proteins General Chemistry Mass spectrometry Proteomics Tandem mass spectrometry Biochemistry Article Molecular Weight Cerebrospinal fluid Liquid chromatography–mass spectrometry Tandem Mass Spectrometry Humans Choroid plexus Biomarkers |
| Zdroj: | Journal of proteome research. 11(6) |
| ISSN: | 1535-3907 |
| Popis: | The cerebrospinal fluid (CSF) is produced in the brain by cells in the choroid plexus at a rate of 500mL/day. It is the only body fluid in direct contact with the brain. Thus, any changes in the CSF composition will reflect pathological processes and make CSF a potential source of biomarkers for different disease states. Proteomics offers a comprehensive view of the proteins found in CSF. In this study, we use a recently developed non-gel based method of sample preparation of CSF followed by liquid chromatography high accuracy mass spectrometry (LC-MS) for MS and MS/MS analyses, allowing unambiguous identification of peptides/proteins. Gel-eluted liquid fraction entrapment electrophoresis (Gelfree) is used to separate a CSF complex protein mixture in 12 user-selectable liquid-phase molecular weight fractions. Using this high throughput workflow we have been able to separate CSF intact proteins over a broad mass range 3.5 kDa-100 kDa with high resolution between 15 kDa and 100 kDa in 2 hours and 40 min. We have completely eliminated albumin and were able to interrogate the low abundance CSF proteins in a highly reproducible manner from different CSF samples in the same time. Using LC-MS as a downstream analysis, we identified 368 proteins using MidiTrap G-10 desalting columns and 166 proteins (including 57 unique proteins) using Zeba spin columns with 5% false discovery rate (FDR). Prostaglandin D2 synthase, Chromogranin A, Apolipoprotein E, Chromogranin B, Secretogranin III, Cystatin C, VGF nerve growth factor, Cadherin 2 are a few of the proteins that were characterized. The Gelfree-LC-MS is a robust method for the analysis of the human proteome that we will use to develop biomarkers for several neurodegenerative diseases and to quantitate these markers using multiple reaction monitoring. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|