Proteomics | SDS PAGE | Applications of Proteomics | Biology Bug

What is Proteomics?

Proteomics is the branch of molecular biology which is the large scale study of proteomes. But what is proteome? A proteome is set of proteins produced in an organism, a system or biological context. Proteome can be studied in specific organism (human, rat) or can be studied in specific organ (liver, pancreas). The proteome is not constant because it differs from cell to cell and changes over a period of time.

Importance of proteome analysis

Proteins are not only the important macromolecules in our body but also the most common effectors of disease pathogenesis and determinants of treatment response. But analysis of proteins is technically challenging and hence we face troubles to develop effective therapeutics. However analysis of DNA and RNA have some role in the prediction of protein function but these results do not always correlate with protein functions. Proteomics help us to overcome these challenges and also in the prediction of protein functions at different levels like post translational modification, domains, motif etc. Despite the inherent limitations of proteomic methodologies, as many as 115 protein based assays have been approved for use by regulatory agencies and commercialize with the great success.

SDS-PAGE or Gel elctrophoresis

Gel based proteomic is the most popular and versatile method of global protein separation and quantification. This is mature approach to screen the protein expression at the large scale. Based on two biochemical characteristics of proteins, two dimensional electrophoresis combines isoelectric focusing which separates proteins according to their isoelectric point and SDS PAGE which separates them further according to their molecular mass. The next typical steps of flow of gel based proteomics are spots visualization and evaluation, expression analysis and finally the protein identification by mass spectrometry. For the study of differentially expressed proteins, two dimensional electrophoresis allows simultaneously to detect, quantify and compare up to thousand protein spots isoform including post translational modifications in the same gel and in a wide range of biological systems.

First dimension

Proteins are amphoteric molecules i.e. they carry both positive and negative charge; hence the net charge on proteins is zero depending upon their amino acid composition. The isoelectric point of protein is the specific pH at which the net charge of protein is zero. Proteins are positively charged at pH values below their isoelectric point and are negatively charged at pH values above their isoelectric point. IEF is an electrophoretic separation based on this specific characteristics of proteins.

Basically the first dimension of two dimensional electrophoresis is achieved with a strip. It is a dry gel that is formed by the polymerization of acrylamide monomers, linked by bis-acrylamide with molecules of covalently linked immobilin, immobilins are chemical components that are derived from  acrylamide and have additional ionizable non amphoteric functions. Immobilins of various pKa can create am immobilized pH gradient inside the gel.

The strip acrylamide gels are dried and cast on a plastic backing. They are rehydrated in a solution containing a pI- corresponding cocktail of carrier ampholytes and with the correct amount of the proteins in the solubilization buffer. The carrier ampholytes are amphoteric molecules with high buffering capacity near their pI.

When an electric field is applied, the negatively charged molecules (proteins and ampholytes) move towards the anode (red electrode) and the positively charged molecules move towards cathode (black electrode). When the proteins are aligned according to their isoelectric point, the global net charge is zero and the protein is unable to move and is then focused. Focusing is achieved with a dedicated apparatus that is able to deliver up to 8000-10000 V, but with a limitation in current intensity to reduce heat.

The equilibration step is critical for 2DE. In this step the strips are with sodium dodecyl sulfate (SDS), an anionic detergent that can denature proteins and form a negatively charged protein complex. The amount of SDS bound to a protein is directly proportional to the mass of protein. Thus, protein that are completely covered by negative charges are separated on the basis of molecular mass.

Second dimension

The SDS denatured and reduced proteins are separated according to an apparent molecular weight, in comparison with a molecular weight maker. Equilibrated strips are embedded with 1% low melting point agarose in TRIS/Glycine/ SDS running buffer and 0.01% bromophenol blue on the top of second dimension gel. When the bromophenol blue migration front reaches the bottom of the gel, the second dimension is finished and the acrylamide gel can be removed from the glass plates.

Application of Proteomics

Mining

Expression profiling

Network mapping

Protein modification

Mining

It is a process of analysing and identifying all the protein samples. Mining is one of the ultimate excercise in proteomics where one simply resolves proteins to the greatest extent possible. It uses MS with associated database and software tools to identify what exactly is found. The several approaches of mining offer the ability to confirm

Expression profiling

Protein Expression profiling is identification of proteins at different level of stages of organsims or cells eg. Development or disease state. Also to analyze the expression due to some genetic, chemical or physical stimulus eg drug. Expression profiling is actually a specialzed form of mining. It is used especially in the cases where two different stages are compared to see which proteins are expressing differently. This technique is used to detect the potential targets for drug therapy and disease.

Protein network mapping

It is an approach to determine how exactly proteins react with each other in a living system. Protein carry out their function in close association with other proteins. They involve in signal transduction, complex biosynthetic and degradation pathways. Most of the protein protein interaction has been studied in vitro. Then why network mapping is important? Proteomic approaches offer the opportunity to characterize more complex network through creative pairing of affinity capture techniques with analytical proteomics methods. They used to identify the components of multiprotein complexes. Multiple complexes are involved in point to point signal transduction pathways in cells. This technique helps to understand all the components in single pathway.

Protein modifications

Another application of proteomics analysis is to identify how and where the proteins are modified. There are many common post translational modifications which can govern the structure, function and turnover of protein. Also many chemicals, environmental factors or drugs can influence the modification of protein. These modified proteins can be detected with antibiodies but the precise sequence sites of specific modification are not known. Proteomics approaches offer the best  means of establishing both the nature and the sequence of posttranslational modifications. These approaches will provide of chemical modifications in domain.

External links

  • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2873371/
  • https://academic.oup.com/hmg/article/25/R2/R182/2198199
  • https://www.ebi.ac.uk/training/online/course/proteomics-introduction-ebi-resources/what-proteomics

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