LECTURE-06-PPT-v2 - NPTELnptel.ac.in/courses/102101007/downloads/PPT/LEC-06-PPT.pdfL. Hagel 2001 IIT...

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Dr. Sanjeeva Srivastava IIT Bombay

IIT Bombay Proteomics Course NPTEL 2

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Chromatography

SEC

IEX

NPC

HIC RP-‐HPLC

HP-‐HILIC

HP-‐IMAC

IIT Bombay Proteomics Course NPTEL

•  Techniques which separate proteins rely on: •  Differential solubility of proteins

•  Size of proteins

•  Charge on proteins

•  Affinity for ligands

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•  Separation of proteins over a bed of appropriate material

•  The material used to pack a column is called matrix/resin, which is usually beads

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•  Chromatography involves four major components: - sample introduction, mobile phase, stationary phase,

detector

•  Chromatography requires selection of matrix - based on bead shape, size, porosity, charge etc.


•  Matrix/resin: usually beads

•  With/without attached chemical groups •  Binding/interaction of proteins with column matrix

is an important feature of chromatography

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L. Hagel 2001


•  Size exclusion chromatography •  according to size

•  Small size molecules retained longer by gel filtration systems

•  Larger protein molecules elute first

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Packed gel column

Chromatography column


Unpurified protein mixture

Sample loading

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Direction of flow

Mobile phase (salt solution)

Mobile phase reservoir


Sample collection

Sample collection

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•  Proteins separated based on charge difference

•  Varying amounts of positive/ negative amino acids

•  pH influences net charge on proteins

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Cellulose Or

Agarose

Cellulose Or

Agarose

Carboxymethyl (CM) Diethylaminoethyl (DEAE)


Chromatography column Packed ion exchange column

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Direction of flow


Mobile phase (buffer solution 1)


Direction of flow


Mobile phase (buffer solution 2)

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Sample collection


Williams A, Frasca V, 2001.

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•  Based on affinity of protein to other molecules

•  Metal chelation widely used in purification of recombinant proteins

•  substrates, products, cofactors, antibodies, metal •  Matrix beads are chemically coupled to ligand

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Chromatography column Column packed with

derivatized resin

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Unpurified protein mixture

Affinity column

Sample loading


Direction of flow


Mobile phase

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Ligand solution

Column elution


Fusion partner Ligand Elution

Protein A IgG Low pH

ABP HSA Low pH

His6 Ni (Metal chelator) Imidazole/ low pH

GST Glutathione Glutathione (reduced)

MBP Amylose Maltose

FLAG M1/M2 Ab EDTA/ Low pH

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1. Load on Ni2+ column

Crude protein extract containing His-tagged protein

Washing removes all proteins except His-tagged proteins

Protein elution 100-500 mM imidazole 2. Wash 20-50

mM imidazole


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•  Separate mixture components on basis of differences in affinity for stationary & mobile phase

•  Removes undesired impurities & concentrates diluted samples

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Solvent tray HPLC Pump

Mobile phase

Pump inlet

Pump outlet – mixed solvents

Solvent bo;les

Flow adjustment valves

Solvent flow to pump

Ardrey 2003


•  Based upon hydrophobic binding interaction: •  peptides/proteins (mobile phase) •  immobilized hydrophobic ligand (stationary phase)

•  RP is used with ESI

32 MS

HPLC pump

B A Mobile phases

Injector RP

column A buffer (0.1% Formic acid, 5% ACN)

B buffer (0.1% Formic acid, 80% ACN)

Desalt and

separate

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•  Silica based cation exchange stationary phase •  Sulfonic acid cation-based exchange ligand •  Ligand covalently bound to polymer coated silica

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Strong ca*on exchanger (SCX) Reverse phase column (RPC)

Sulphonic acid groups Long hydrophobic alipha*c chains

7Separation by charge Separation by hydrophobicity

RP CE

SEC CE

IMAC RP Avidin RP

SCX RP

SEC RP


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•  Hagel L. 2001. Current Protocols in Molecular Biology. UNIT 10.9 Gel-Filtration Chromatography. DOI: 10.1002/0471142727.mb1009s44 •  Ardrey R E. 2003. Liquid Chromatography - Mass Spectrometry: An Introduction. Chapter 2. Liquid Chromatography. DOI: 10.1002/0470867299.ch2. •  Williams A, Frasca V, 2001. UNIT 8.2 Ion-Exchange Chromatography. Current Protocols in Protein Science •  Cravatt BF, Simon GM, Yates JR, The biological impact of mass-spectrometry-based proteomics. Nature 450, 991-1000 (13 December 2007) •  Berg J., Tmyoczko J. & Stryer L., Biochemitry fifth ed., W. H. Freeman & company, 2002. ISBN: 0716746840. • Nelson D. & Cox M.,Lehninger, Principles of Biochemistry fourth ed., W. H. Freeman and company, 2004. ISBN: 023022699X. • Voet D. & Voet J., Biochemistry fourth ed., Wiley, 2000. ISBN: 047158651X. •  Issaq et al. Methods for fractionation, separation and profiling of proteins and peptides. Electrophoresis. Volume 23, Issue 17, pages 3048–3061, No. 17 September 2002

LECTURE-06-PPT-v2 - NPTELnptel.ac.in/courses/102101007/downloads/PPT/LEC-06-PPT.pdfL. Hagel 2001 IIT...

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Transcript of LECTURE-06-PPT-v2 - NPTELnptel.ac.in/courses/102101007/downloads/PPT/LEC-06-PPT.pdfL. Hagel 2001 IIT...