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2D Micro-HPLC System for Proteome Analysis
Proteome analysis with greater productivity and performance
The high-performance 2D micro-HPLC system can perform separation of complex peptide mixtures with a level of sensitivity and resolution that meets or exceeds present-day requirements. Combining cation exchange chromatography in the first dimension and capillary reversed-phase chromatography in the second dimension, using 6 trap columns, allows for the various separation conditions to be optimized independently. This enables separation analysis for large numbers of trace peptides from digests therefore proving to be an extremely effective tool for the discovery of unknown proteins with low expression levels. When this system is coupled with a high-performance mass spectrometer with an electrospray ionization interface (ESI), unparalleled performance and sensitivity can be achieved.
Features
1. It is possible to analyze proteins that cannot be analyzed with 2D gel electrophoresis.
Highly ionic proteins and hydrophobic proteins can be analyzed on-line.
2. Automatic analysis with high resolution and sensitivity is possible for proteins with low expression levels.
Protein digests can be analyzed automatically by simply placing them into an autosampler. Assay sensitivity enables trace peptide analysis sometimes not detected on 2-D gels.
3. Stable analysis is ensured by on-line sample trapping and automatic desalination followed by RPHPLC.
Sample from the cation exchange column is trapped on 6 mini trap columns (polymer packed reversed-phase columns with a retention power equivalent to C4) for desalination followed by reversed-phase analysis. This method ensures stable, high-precision analysis.
Analysis Example
The analysis results for a trypsin digestion of a mixture of 6 types of protein (1 pmol each) are shown in Fig. 3. For comparison, the top chromatogram shows the analysis results obtained with a 1D LC-MS chromatogram, while the bottom overlaid chromatograms show the results of analyzing each fraction in the first dimension with a reversed-phase LC-MS in the second dimension. It can be seen that the level of resolution for peptides is greatly improved by using this system.
The salt used for elution on the primary side is ammonium sulfate; the concentrations for each digest are 1 mM, 10 mM, 20 mM, 30 mM, 50 mM, and 99 mM respectively. The flowrates were set to 80 uL/min (internal diameter of column: 1 mm) and 10 uL/min (internal diameter of column: 300 mm) in the first and second dimensions, respectively.
Shimadzu Corporation is participating in the joint development of multi-dimensional HPLC systems for proteome analysis based on a CRADA concluded between Shimadzu and the NIH (NIMH).
CRADA:
Cooperative Research And Development Agreement, (An official joint research program formed between a research institute controlled by the US Department of Health and Human Services and a corporation or other research institute)
NIMH:
National Institute of Mental Health.