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doi:10.1016/j.chroma.2005.12.069 
Published by Elsevier B.V.
Proteomic analysis of rat plasma by two-dimensional liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry
Available online 2 February 2006.
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Abstract
The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers therefore requires either the specific depletion of high abundance proteins using immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe a two-dimensional (2D) liquid chromatography separation method for the fractionation of rat plasma. In the first dimension proteins were separated by chromatofocusing according to their isoelectric point (pI). In the second dimension, proteins were further fractionated by non-porous, reversed-phase chromatography according to their hydrophobicity. The data from both separations was displayed as a 2D protein expression map of pI versus retention time (relative hydrophobicity). Both separations were carried out on the ProteomeLab PF 2D system (Beckman Coulter), an instrument platform that provides a high degree of automation and real-time monitoring of the separation process. The reproducibility of the first-dimension separation was evaluated in terms of pH gradient formation. The second-dimension separation was evaluated in terms of peak retention times on the reversed-phase column. We found in four consecutive chromatofocusing separations that the pH gradient differed by less than 0.2 pH units at any time during the elution step. Second dimension retention times of peaks from identical pI fractions differed by less than 7 s in six consecutive separations. Each 2D separation generated a total of 540 fractions which were analyzed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). We detected approximately 275 peptides and proteins with molecular masses ranging from 3 to 225 kDa. Most fractions were found to contain multiple low and high molecular weight proteins. Differential display of 2D protein expression maps from retinol-sufficient and -deficient rat plasma samples identified a fraction with several proteins that appeared to be down-regulated in the vitamin A-deficient animal. Quantitative proteomic analysis of complex samples such as plasma is still a difficult task. We discuss the potential of this approach for biomarker discovery and address the experimental challenges that remain.
Keywords: Two-dimensional chromatography; Chromatofocusing; Reversed-phase HPLC; MALDI; Plasma; Retinol; Vitamin A
Article Outline
- 1. Introduction
- 2. Experimental
- 2.1. Reagents
- 2.2. Instrumentation
- 2.3. Chromatographic methods
- 2.4. Mass spectrometric methods
- 2.5. Animal procedures
- 2.6. Statistical analysis
- 3. Results and discussion
- 3.1. Two-dimensional liquid separation of rat plasma by chromatofocusing and non-porous, reversed-phase chromatography
- 3.2. MALDI-TOF MS analysis of second dimension fractions
- 3.3. Differential protein expression profiling of plasma samples from retinol-sufficient and -deficient rats by 2D liquid phase separation
- 4. Conclusion
- Acknowledgements
- References
0.3 pH units were collected and are displayed in the x-axis. Second dimension elution time is indicated in the y-axis and corresponds to increasing acetonitrile concentration (28.5–71.5% solvent B). Color scale represents the relative intensity of each band based on UV detection at 214 nm (red = most intense, purple = background).
) were spotted on target plate, mixed with matrix and analyzed by MALDI-TOF MS. Insert shows second dimension chromatogram of reversed phase separation. Peak marked with 
