Robust Two-Dimensional Separation of Intact Proteins for Bottom-Up Tandem Mass Spectrometry of the Human CSF Proteome
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- 作者：Adriana Bora ; Carol Anderson ; Muznabanu Bachani ; Avindra Nath ; Robert J. Cotter
- 刊名：Journal of Proteome Research
- 出版年：2012
- 出版时间：June 1, 2012
- 年：2012
- 卷：11
- 期：6
- 页码：3143-3149
- 全文大小：294K
- 年卷期：v.11,no.6(June 1, 2012)
- ISSN：1535-3907
文摘
Cerebrospinal fluid (CSF) is produced in the brain by cells in the choroid plexus at a rate of 500 mL/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 nongel based method of sample preparation of CSF followed by liquid chromatography鈥揾igh 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鈥?00 kDa) with high resolution (between 15 and 100 kDa) in 2 h 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 at 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 a 5% false discovery rate (FDR). Prostaglandin D2 synthase, Chromogranin A, Apolipoprotein E, Chromogranin B, Secretogranin III, Cystatin C, VGF nerve growth factor, and Cadherin 2 are a few of the proteins that were characterized. 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.