Introduction To Proteomics And Technologies Biology Essay

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Introduction to Proteomics

Proteome is the set of all the proteins encoded by the genome; the study of the proteome is called proteomics. Proteomics also assesses all proteins isoforms, structures, functions, modifications, localization and interactions between them. Encoded proteins carry out most biological functions, and to understand how cells work, one must study what proteins are present, how they interact with each other and what they do (Marte 2003).

As a result of alternative splicing, Post translational modifications, like sugar added, peptides clipped off or phosphorylation after translation, the same gene can generate multiple different proteins products (figure 1). Therefore, the organism's proteome is more complex than its genome.

The promise of proteomics is the precise definition of the function of every protein in the cell, and how that function changes in different environmental conditions, with different modification states of the protein, in different cellular locales, and with different interacting partners (Phizicky 2003)

Technologies that are use to do Proteomics

Two-dimensional gel electrophoresis

This is a gel-based method, which attempts to separate all the proteins of a particular cell or tissue. First, we separate the proteins according to the charge and then, according to their molecular weights. The principle is illustrated in Figures 1b, 2.

The 2DE gels are then digitized and qualitatively and quantitatively analyzed with specialized software programs. Some of the advantages of 2DE are, high-resolution results, based on two independent protein characteristics (charge and molecular weight), a huge number of proteins can be visualized simultaneously on a single 2DE gel and the equipment is relatively inexpensive. Also 2DE can distinguish between different protein isoforms a

Figure 2. Principle of the two-dimensional electrophoretic separation of intact proteins. The sample is loaded onto IEF to IPG strip, where proteins are separated according to their isoelectric points (pI). Next, the strip is loaded on SDS-PAGE, where the proteins are separated according to their molecular weights (MW). Visualization of protein spots is achieved via chemical staining methods. Individual spots can be further excised and analyzed by mass spectrometry (Zybailov, Washburn 2006).

nd post translational modifications. 2DE is an easy to learn technique, because most scientists are familiar with gel electrophoresis. On the other hand, the disadvantages of 2DE are that it is labor intensive, and some of the gel spots can overlap, and the individual protein identification can give incorrect results.

Another weakness, is that it is a denaturing technique, therefore it is not suitable for the analysis of protein complexes and interactions, it is also limited by the resolution of the spot and therefore to the visualization methods. An additional drawback is that it is not possible to analyze the entire proteome. Proteins in a 2DE gel represent only a portion of all the proteins that are normally present in a sample.

Some proteins with specific properties are difficult to analyze, such as: very small and very large proteins, alkaline proteins, hydrophobic proteins, and membrane proteins.

Finally 2DE is a labor-intensive technique with a relatively low throughput, not adequate for projects that involve screening of a large number of clinical samples.

Mass Spectrometry


Mass Increase (Da)














162Mass spectrometry (MS), which is used for determining masses of molecules, is ideal for large scale and accurate analysis of post-translational amino acids modifications (Mann 2009). Each change adds a specific increase in the mass of the modified amino acids (Table 1).

Table 1. Mass increases resulting from common post translational modifications (Murray 2003).The principle behind MS, consist of ionizing the molecules to charge them and measure their mass to charge ratios.

Figure 3. Matrix assisted laser desorption(MALDI)-TOF Mass Spectrometry. (1) The protein sample, embedded in an appropriate matrix, is ionized by the application of a laser beam. (2) An electrical field accelerates the ions formed through the flight tube toward the detector. (3) The lightest ions arrive first. (4) The ionizing laser pulse also triggers a clock that measures the time of flight (TOF) for the ions ( Watson, Sparkman 2007 ).In a MS procedure, we vaporized a sample in a vacuum chamber, under conditions where protonation can occur, imparting positive charge to the molecules. Then ions are separated in an electrical field and the cations move through a magnetic field onto a detector, the lightest ions arrive first (Figure 3). Therefore, the time required for each particle to reach the detector, will be inversely proportionate to their mass.

MS data is represented in various ways; the most used is the mass spectrum, intensity vs. mass/charge shown in figure 4. Mass chromatogram, is another type of data representation, where x-axis represent time and y-axis the intensity.

Figure 4. MALDI-TOF Mass Spectrum of Insulin and β -lactoglobulin. A mixture of 5 pmol each of insulin (I) and β -lactoglobulin (L) was ionized by MALDI, which produces predominately singly charged molecular ions from peptides and proteins (I + H+ for insulin and L + H+ for lactoglobulin). However, molecules with multiple charges as well as small quantities of a singly charged dimer of insulin, (2 I + H)+, also are produced. ( Watson, Sparkman 2007 )

Results of MS so far, have been more qualitative than quantitative, to have a quantitative result we can use a label free quantification approach, a combination of 2DE and MS, or employ differential stable isotope labeling to create a specific mass tag that can be recognized by a mass spectrometer and at the same time provide the basis for quantification (figure 5).

Figure 5. Schematic representation of methods for stable isotope protein labeling for quantitative proteomics.(Aebersold, Mann 2003)

Using ion traps analyzers for quantitative MS, has proven to be a robust, sensitive and inexpensive technique, but one of the disadvantage is their relatively low mass accuracy.

In the future, quantitative methods based on stable isotope labeling are likely to revolutionize the study of stable or transient interactions and interactions dependent on post-translational modifications (Aebersold, Mann 2003).