By Ingvar Eidhammer; et al
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Extra info for Computational methods for mass spectrometry proteomics
Com/proteomics/ 3 Protein sample Protein digestion Protein digestion Peptide sample Peptide fraction MS experiment Peptide fraction MS experiment Peptide separation An important part of the identification process is protein digestion: the cleaving of proteins into peptides. Protein cleavage can be performed chemically or enzymatically, with enzymatic cleavage the most often used approach. The enzyme(s) that perform(s) the digestion are called proteases, and these are found in all organisms. For example, in the intestine, proteins derived from food are cleaved into small pieces to achieve an efficient absorption, while inside individual cells proteins are continuously degraded by proteases as a part of regulatory mechanisms.
No staining technique will stain all types of proteins in proportion to the amount of protein present, implying that they are unsuited for accurate quantification inside a gel. 5 shows a 2D gel. 3 37 Problems There are several problems with 2D SDS-PAGE, as follows: • Not all proteins will appear on the gel. 4; – proteins with an isoelectric point below 3 or above 10; – proteins with a mass below 8 kDa, or above 150–200 kDa; – proteins of low abundance. The hydrophobic proteins tend to form complexes and precipitate before they can be loaded on the pH gradient, while the proteins with extreme pI usually fall off the edges of the pH strip.
The pI of proteins is usually in the range of 3 to 12, but the overall majority are between 4 and 7. The strips typically have either a very broad range (for example, pH 3–10), or a very narrow range (for example, pH 4–5). 4 (a) A pH gradient filled initially with the protein sample. Note that the sample may also be applied at one end of the pH gradient. (b) When an electric field is established, the proteins move to the pH equal to their pI values. 3 Separation on mass and isoelectric point – 2D SDS-PAGE The SDS-PAGE or IEF separation methods alone do not have high enough separating power for sufficient resolution of complex protein samples.
Computational methods for mass spectrometry proteomics by Ingvar Eidhammer; et al