Robust Two-Dimensional Separation of Intact Proteins for Bottom-Up Tandem Mass Spectrometry of the Human CSF Proteome

Abstract

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.

In-depth Proteomic Analysis of Mouse Cochlear Sensory Epithelium by Mass Spectrometry

“…This technique offers separation reproducibility, sample enrichment, high protein recovery, and reduces the distribution of high abundant proteins in a complex protein sample. 28 Unlike traditional 2D-PAGE, where proteins are extracted from gel spots, in GELFrEE the protein mixture is fractionated in liquid-phase, which allows for more efficient digestion and higher sample recovery. A silver-stained gel was prepared to visualize the results from GELFrEE fractionation prior to multi-enzyme digestion and LC-MS/MS analysis (Supporting Information Figure 3).…”

Efficient synthesis of molecularly imprinted polymers with bio‐recognition sites for the selective separation of bovine hemoglobin

“…In recent years, to study the characteristics of the protein in various scales such as the protein translation, protein expression and protein–protein interaction has attracted increasing attentions. Proteins, like antibodies and enzymes, are usually employed as recognition elements for diagnosis and disease therapeutics . However, these kinds of proteins often present at low‐abundance levels in complex matrix and hard to analyze them directly.…”

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

“…There are two commonly used MS proteome analysis approaches, shotgun and bottom-up proteomics. In shotgun proteomics, a mixture of intact proteins is enzymatically digested and separated using multidimensional chromatography with strong cation-exchange chromatography (SCX) followed by reversed-phase liquid chromatography (RPLC) 14,15 . The separated peptides are subjected to tandem MS and database searching 15 .…”

“…In shotgun proteomics, a mixture of intact proteins is enzymatically digested and separated using multidimensional chromatography with strong cation-exchange chromatography (SCX) followed by reversed-phase liquid chromatography (RPLC) 14,15 . The separated peptides are subjected to tandem MS and database searching 15 . A major advantage of this technique is that thousands of proteins can be identified in a single analysis and the technique is better suited to membrane proteins.…”