title: Robust two-dimensional separation of intact proteins for bottom-up tandem mass spectrometry of the human CSF proteome.
abstract: 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-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 (3.5-100 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.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine , Baltimore, Maryland, United States.
authors.forename: Adriana
authors.initials: A
authors.lastname: Bora
authors.forename: Carol
authors.initials: C
authors.lastname: Anderson
authors.forename: Muznabanu
authors.initials: M
authors.lastname: Bachani
authors.forename: Avindra
authors.initials: A
authors.lastname: Nath
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Cotter
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002556:Cerebrospinal Fluid Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D056148:Chromatography, Reverse-Phase; D006801:Humans; D008970:Molecular Weight; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D015415:Biomarkers; D002556:Cerebrospinal Fluid Proteins; D020543:Proteome