Last modified on 11 July 2012, at 18:37

Proteomics/Emerging and Miscellaneous Proteomics Technologies


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Protein Separations - Centrifugation
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Emerging and Miscellaneous Proteomics Technologies
Protein Identification - Mass Spectrometry
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Protein Identification - Mass Spectrometry

 

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Introduction
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Emerging and Miscellaneous Technologies in Proteomics

This part of the book will be an area where proteomics techniques that have been newly developed will be discussed. Techniques that do not currently fit into any other part of the book can also be added to this page. As chapters or sections are added elsewhere that discuss these techniques in the context of a greater proteomics problem, the information on this page will be moved to those pages.

X-Ray Tomography

Description and Discussion of X-ray Tomography.

A new branch of X-ray microscopy is being used in proteomics analysis. This is called X-ray Tomography. This method uses projected images to calculate and reconstruct a 3D object. This technology is being used in proteomics to determine the location of labeled proteins or large complexes within a cell. This technique can also be used in conjunction with images of cells from light based microscopes to help identify where a protein is located and how this location factors in to its function and identification.

Introduction to Proteoinformatics

Proteoinformatics is the use of bioinformatics and computational biology techniques solely within the realm of protein identification and proteomics. Proteoinformatics is currently in its infancy and the largest work being done is on standardizing databases and data submission. Other proteoinformatic work is being done on the image analysis of 2D gels and other images in proteomics used to help identify and annotate proteins in the proteome.

Protein Identification Database

What are Protein Identification Databases?

Protein Identifications Databases such as ProFound at Rockefeller University and Protein Prospector at the UCSF Mass Spectrometry Facility are used to help identify proteins found with proteomics techniques such as mass spectrometry. Digestion of proteins into peptide fragments allows each protein to break apart in a different way, resulting in a unique peptide fingerprint that can be used to identify the protein. The masses of these fragments as well as the molecular weights and isoelectric points are what is stored in many of these databases. This data can be used to perform high-throughput protein identification.

The Future of Protein Identification Databases

In order to continue advancing the cause of mapping the human proteome, international databases need to be established which integrate both transcriptome and proteome data. The Human Proteome Organization is currently working on establishing a defined standard for data submission and annotation for the many different proteomics techniques currently used to identify and annotate proteins.

New Techniques in Image Analysis

According to the Image Analysis Wikipedia page, "Image analysis is the extraction of meaningful information from images." In terms of Proteomics, image analysis can be used to compare different images generated using proteomics techniques, such as 2D-PAGE gel images. New programs are being developed that will help to optomize and automate the process of locating a protein spot between two gel images in order to identify the differences between 2D-PAGE gels. Other programs can be used to help clean up and remove variability between these images as well.

Laser Capture Microdissection

Diagram of Laser Capture Microdissection

Laser capture microdissection or LCM is a process that isolates and removes distinct populations from a tissue. This will facilitate the comparison of diseased tissue with normal tissue from an organism.

In LCM, an infrared laser beam melts a thermosensitive polymer film that traps a specific group of cells. This polymer film is then extracted and moved to a test tube where an extraction buffer is used to remove the groups of cells for more advanced proteomics analysis such as 2D-PAGE, Ion Chromatography, etc. This technology will become more useful as systems with higher sensitivity for analysis of smaller amounts of tissue become developed and realized.

Proteomic Complex Detection using Sedimentation

Approaches such as TAP tagging, which require the addition of fusion proteins, can interfere with protein interactions that would have normally occurred. Many times it takes a great deal of work to express these tagged proteins, so this technique is used to give evidence that there is a stable protein complex detected early on in the proteomics experiment before more laborious approaches are used to isolate and identify the protein complexes of interest. Issues also occur in MS and 2D Gel processes where one cannot be sure that a portion of a gel spot is the desired protein because multiple proteins could be traveling together in that spot as a complex.

This is where proteomic complex detection using sedimentation (ProCoDeS) is applicable. ProCoDeS is a technique for the high-throughput identification of both soluble and membrane proteins that are found in stable complexes. Relative sizes of protein complexes are estimated via their sedimentation in a gradient. In this case a rate zonal gradient or RZG is used to better estimate the relative size of protein complexes. The distribution of a protein of interest in this sedimentation can be detected using classic techniques such as Western Blotting or newer techniques such as ICAT. This can be done for a large number of proteins. Thus, ProCoDeS can be used to identify stable protein complexes. ProCoDeS is especially well suited for the screening of unrefined cellular material to help find new proteins that cannot be discovered because they exist in protein complexes such as proteins found in protein membranes.


Next Chapter: Protein Identification - Mass Spectrometry

References

  1. Hartman, N. T., et al. "Proteomic Complex Detection Using Sedimentation" Anal. Chem., 79, 5, 2078 - 2083, 2007.
  2. NCT Proteomics Group "Emerging Technologies" National Institutes of Environmental Health Sciences
  3. NCT Proteomics Group "ProteoInformatics" National Institutes of Environmental Health Sciences
  4. "Wikipedia: Image Analysis
  5. "Wikipedia: Proteomics
  6. "Wikipedia: X-ray Tomography