Question

Give a detailed account of three different methods used in proteomics ?

Answer 1

Proteomics describes the global analysis of proteome, which can be defined has as the protein content of a cell, a tissue or an entire organism in a defined state. Since the inception of this field 30 years ago, the complete characterization of all proteins has been its fundamental goal.

SDS-PAGE

Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) is an electrophoretic technique widely used in biotechnology, biochemistry, molecular biology, forensic science and other life science laboratories. In SDS-PAGE, proteins are separated in a palyacrylamide gel based on their molecular weight. Proteins are amphoteric molecules, i.e. they have both positive and negative charges. To make them move in a single direction, a uniform negative charge is created on them. When the proteins are mixed with SDS, they acquire a net negative charge.

SDS is a detergent having a negative charge, therefore it is an anionic detergent. SDS denatures the native proteins by disturbing the non-covalent forces. The non-covalent forces include hydrogen-bonding, hydrophobic and ionic interactions which are responsible for the three-dimensional structure of a native protein. SDS also gives a uniform net negative charge to the protein molecules. The denatured linear protein molecules are loaded onto the polyacrylamide gel (PAGE) which is made by polymerizing the acrylamide monomers. PAGE is prepared using acrylamide, bisacrylamide, TEMED, ammonium persulfate and Tris-HCl buffer. PAGE has two phases: a stacking gel and a separating gel. Under an applied electric field, the stacking gel concentrates the SDS-loaded linear protein molecules while the separating gel separates the proteins on the basis of molecular weight. After the run, PAGE gel is placed in a Coomassive Brilliant Blue R250 dye solution for staining for a few hours and is de-stained to visualize the separated protein molecules as bands.

2D protein electrophoresis
Nowadays the two-dimensional (2D) protein electrophoresis is a method of choice for protein separation. This method enables to distinguish up to 10 000 proteins. The method is based on two distinct physical and chemical features of proteins: first, the proteins are separated according to their isoelectric point (pI). Isoelectric point is such a pH value, where the overall protein charge equals to zero. This can be obtained by creating a pH gradient in the gel where protein is loaded and electric current is applied. Proteins then migrate towards cathode or anode according to their total charge up to the point where the gel pH equals pI of a given protein. After the separation in the first direction, common electrophoresis on a polyacrylamide gel (PAGE) is applied, but the electric current is applied perpendicular to the original orientation of electrodes. Proteins then migrate in the second direction through gel only according to their size. After both phases of 2D electrophoresis, it is necessary to visualize proteins by one of the staining or labeling methods (chemical or radioactive). The resulting "maps" of proteins can be compared for example in between the experimental and control sample or among the samples from patients with specific disease and their healthy controls and thus identify differentially expressed proteins that can be linked with the pathogenesis of the studied disease. The identity of differentially expressed proteins is verified after "cutting out" the area of the gel with given difference and subsequent analysis using mass spectrometry.

Mass
Mass spectrometry is a method that enables precise measurement of molecular weight of a broad spectrum of substances. As the studied substance has to be intact in gas phase, the use of mass spectrometry for protein analysis (but also of polysaccharides and oligonucleotides) was only enabled by development of "soft" ionization techniques of mass spectrometry, like matrix assisted laser detection of desorption/ionization (MALDI) and electrospray ionization (ESI). Protein identification is generally performed in two ways:
1. Protein is digested by trypsin or by other proteolytic enzyme to smaller peptides and their precise molecular weights are measured using MS. The spectrum of those molecular weights is then compared with theoretical spectra that are calculated from protein sequences from available databases (using bioinformatics tools).
2. Tandem MS enables to choose the peptide which is then fragmented by the collision with inert gas. The fragmentation pattern gives either full of partial information about protein sequence that is subjected to the search in databases.
Apart of protein identification, MS is a priceless tool for protein posttranslational modification analysis, because it enables to localize given modifications within the protein and also helps to find out the nature of such modification.