Proteomics - KOCWcontents.kocw.net/.../korea_sejong/baekseungpil/04.pdf · •GPEC : Gradient...

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Department of Biotechnology and Bioinformatics Laboratory of Nano-Biotechnology and Artificial Bioengineering 1 Proteomics Chapter 2. strategies for protein separation

Transcript of Proteomics - KOCWcontents.kocw.net/.../korea_sejong/baekseungpil/04.pdf · •GPEC : Gradient...

Page 1: Proteomics - KOCWcontents.kocw.net/.../korea_sejong/baekseungpil/04.pdf · •GPEC : Gradient polymer elution chromatography . Department of Biotechnology and Bioinformatics Laboratory

Department of Biotechnology and Bioinformatics

Laboratory of Nano-Biotechnology and Artificial Bioengineering

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Proteomics

Chapter 2.

strategies for protein separation Ⅱ

Page 2: Proteomics - KOCWcontents.kocw.net/.../korea_sejong/baekseungpil/04.pdf · •GPEC : Gradient polymer elution chromatography . Department of Biotechnology and Bioinformatics Laboratory

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

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Department of Biotechnology and Bioinformatics

Laboratory of Nano-Biotechnology and Artificial Bioengineering

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Liquid chromatography in proteomics

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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

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Department of Biotechnology and Bioinformatics

Laboratory of Nano-Biotechnology and Artificial Bioengineering

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Affinity chromatography

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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).

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Department of Biotechnology and Bioinformatics

Laboratory of Nano-Biotechnology and Artificial Bioengineering

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(Figure 2.9~11)

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Department of Biotechnology and Bioinformatics

Laboratory of Nano-Biotechnology and Artificial Bioengineering

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Page 9: Proteomics - KOCWcontents.kocw.net/.../korea_sejong/baekseungpil/04.pdf · •GPEC : Gradient polymer elution chromatography . Department of Biotechnology and Bioinformatics Laboratory

Department of Biotechnology and Bioinformatics

Laboratory of Nano-Biotechnology and Artificial Bioengineering

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Ion exchange chromatography

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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).

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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

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Department of Biotechnology and Bioinformatics

Laboratory of Nano-Biotechnology and Artificial Bioengineering

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Reversed-phase (RP-)chromatography

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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).

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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.

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Department of Biotechnology and Bioinformatics

Laboratory of Nano-Biotechnology and Artificial Bioengineering

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Size exclusion chromatography

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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

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Department of Biotechnology and Bioinformatics

Laboratory of Nano-Biotechnology and Artificial Bioengineering

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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