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Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
1
Proteomics
Chapter 2.
strategies for protein separation Ⅱ
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
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Liquid chromatography in proteomics
• Any separation technique that distributes the components of a mixture between two phases, a fixed stationary phase and a free-moving mobile phase, is known as chromatography.
• In proteomics, liquid chromatography is used more often than other methods.
– Versatility
– Compatibility with mass spectrometry
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
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Liquid chromatography in proteomics
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
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Affinity chromatography (AC)
• AC partitions proteins or peptides on the basis of their specific ligand-binding affinity.
• We put ligands that are highly selective for particular proteins in the matrix on an affinity column.
• AC methods involve a 2-step elution procedure
– 1st fraction comprises proteins that failed to interact with the affinity matrix
– 2nd fraction comprises all the proteins retained on the column
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
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Affinity chromatography
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
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Ion exchange chromatography (IEC)
• Other than AC, all chromatography methods are non-selective.
• IEC separates proteins according to their charge (Figure 2.10).
• Based on the reversible adsorption of solute molecules to a solid phase that contains charged chemical groups (Table 2.1).
• Produces chromatogram, where x-axis displays elution time and y-axis shows absorption peaks that correspond to individual components of the sample (Figure 2.11).
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
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(Figure 2.9~11)
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
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Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
9
Ion exchange chromatography
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
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Reversed-phase (RP-)chromatography
• RP-Chromatography is similar to IEC, but hydrophobicity is the driving force.
• Reversed-phase resin consists of hydrophobic ligands (Figure 2.12).
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
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• Of all the chromatography techniques used in proteomic, RP-HPLC is the most powerful method and has the highest resolution.
Reversed-phase (RP-)chromatography
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
12
Reversed-phase (RP-)chromatography
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
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Size exclusion chromatography (SEC)
• SEC, also known as gel filtration chromatography is a profiling technique used to separate proteins according to their size (Figure 2.13).
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
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Size exclusion chromatography (SEC)
• The column is packed with inert beads made of a porous compound.
• Small proteins enter the pores and thus take longer to go through the column.
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
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Size exclusion chromatography
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
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Multidimensional liquid
chromatography (Multi-D-LC)
• LC is often used either upstream or downstream of 2DGE to prefractionate samples and to separate tryptic peptides prepared from individual gel spots (Figure 2.9).
• Many of the Limitations of 2DGE are circumvented by LC systems. Multi-D-LC is more diverse and can eventually replace 2DGE.
• LCCC : LC at critical conditions
• GPEC : Gradient polymer elution chromatography
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
17
Multidimensional liquid
chromatography (Multi-D-LC)
Separation mode: LCCC or GPEC SEC Column: RP-C18 or NP Silica gel Styrene/DVB Mobile phase: Isocratic or Gradient THF or DMF
1st dimension 2nd dimension