Proteomics/Protein Separations - Chromatography/Two Dimensional (2D) Chromatography

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Two Dimensional (2D) Chromatography
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Hydrophobic Interaction Chromatography (HIC) Ultra High Performance Liquid Chromatography

Chapter written by: Laura Grell and Alexander Butarbutar
Contact llg3875@rit.edu or nbb3924@rit.edu for contributions
Chapter modified by Kai Burnett and Dalia Ghoneim
Contact kab9783@rit.edu or dxg6098@rit.edu

Two Dimensional (2D) Chromatography[edit]

   Separation of highly complex mixture can be a very difficult task. The mixture can be distributed according to their molecular mass, chemical composition, fuctionality and architecture (Res 5). A single chromatography experiment may be inefficient in separating our proteins of interst. In 2D chromatography, different techniques are essentially combined to achieve a higher degree of separation. This can be done by an offline technique, where the result of one chromatography is injected manually into a second column chromatography or an online method, where the two colums are directly coupled through switches. (Ref 6).

2-D chromatography is considered a complement to 2-D electrophoresis. While 2-D electrophoresis separates proteins based on Isoelectric point and size, 2-D chromatography can use many different combinations of properties for separation. There are many different types of 2D chromatography, these include:

  • Multidimensional Protein Identification Technology (Mud-PIT)

Multidimensional Protein Identification Technology (MudPIT) is a separation technique which utilized two chromatography techniques back to back and can be coupled to silica capillary (Res1). Typically the column consist of strong cation exchanger (SCX) followed by reversed phase (RP) material. One of the advantages of MudPIT is that band broadening are minimized and the capillary can easily be coupled directly with a mass spectrometer. The strategy initially involves denaturing the protein complex into small peptides. The samples are then loaded to strong cation exchanger (SCX) column subsequently followed by reversed phase column. A capillary can be set up to stream line the process with a mass spectrometer. 2D plot image from MudPIT

  • Affinity-Reverse Phase
  • Size Exclusion Chromatography-Reverse Phase
  • Size Exclusion Chromatography-Ion Exchange Chromatography
  • Ion Exchange Chromatography-Reverse Phase

2-D chromatography is growing in popularity (Res 2) as an alternative to 2-D electrophoresis. It is good for separating proteins that have a more extreme range of pIs or a large range of molecular weights. A nice advantage of 2-D chromatography is that it can be coupled to a mass spectrometry device directly. In electrophoresis, the samples must be cut from the gel for retrieval.

An example of a setup utilizing 2D Chromatography can be seen below:
2D Chromatography.gif

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Resources[edit]

  1. Chemical Society Network MudPIT
  2. GE HealthCare - How To Combine Purification Steps || Multidimensional liquid chromatography
  3. The MudPIT Page
  4. Beckman Coulter ProteomeLabTM Protein Fractionation System
  5. Polymer Standards Service A primer in 2-dimensional liquid chromatography

References[edit]

  1. Kislinger T, Emili A. Multidimensional protein identification technology: current status and future prospects. Expert Rev Proteomics. 2005 Jan;2(1):27-39. *
  2. Venkatramani CJ, Zelechonok Y. Two-Dimensional Separations: Concept and Promise J Chromatogr A. 2005 Feb 25;1066(1-2):47-53.
  3. Link AJ, Eng J, et al. Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol. 1999 Jul;17(7):676-82.
  4. Paoletti AC, et al Principles and applications of multidimensional protein identification technology. Expert Rev Proteomics. 2004 Oct;1(3):275-8. *
  5. Takahashi N, et al Automated tandem high-performance liquid chromatographic system for separation of extremely complex peptide mixtures. J Chromatogr. 1985 Jun 19;326:407-18.
  6. Venkatramani CJ, et al Two-dimensional liquid chromatography with mixed mode stationary phases. J Chromatogr A. 2005 Feb 25;1066(1-2):47-53.
  7. Franktisek Svec Two-Dimensional High-Performance Liquid Chromatography *

.* Denotes Free Article