¬†¬†¬†¬†2-D PAGE, as a very important tool for proteomics, is currently the only technique to reveal hundreds or thousands of proteins at a time although it requires manual dexterity and precision and thus not high-throughput technology. Proteins are separated on the basis of charge in the first dimension and molecular mass in the second.
¬†¬†¬†¬†After Cell or tissue samples are solubilized, proteins will be extracted in buffers.
The protein mixture firstly will be separated by isoelectric focusing, the charged proteins migrate in a polyacrylamide gel strip that contains an immobilised pH gradient until they reach the pH at their pI, Then the gel strip will be transferred to polyacrylamide gel containing sodium dodecyl sulphate, and separated on the basis of molecular size. After electrophoresis completed, the resolved proteins are detected by chromophoric staining with coomassie brilliant blue or silver and fluorescent dyes, according to different requirement. Images and different spots are captured by scanning the gels and analyzed by using 2D-image-analysis software, such as PDQuest.
-- 2-D instrument
¬†¬†¬†¬†After samples are ionized and enter Mass Analyzer, the proteins/ peptides will be identified and MW will be determined according to the the difference of mass-to-charge (m/z) ratio. Moreover, the analysis of phosphorylated and glycosylated proteins, ICAT (Isotope Coded Affinity Tages), peptides DE NOVO sequencing, shotgun identification of complex protein digests will be performed with MS technology A MS instrument is composed of injection, ionization source, mass analyzer, ion detector and data analyzer. Matrix-assisted laser desorption/ ionization MS(MALDI-MS) and electrospray ionization MS(ESI-MS) have become main ionization methods in proteins and peptides analysis.
Ionization by MALDI involves proteins suspended or dissolved in solid substance. The substance absorbs energy of the laser that makes the substance vaporize, then it causes the analytes ionize and takes it into the gas phase. The advantage of MALDI-MS are that it can analyze the molecule with large molecular weight and has good tolerance to impurity.
Electrospray ionization (ESI) involves the production of gaseous ions by application of a high potential to a flowing liquid resulting in the formation of a spray of small droplets with solvent-containing analytes. Solvent is removed from the droplet by heat dry gas and strong electric field, then the droplets are gasified and ionized. As the droplet size further decreases they reach a point at which become unstable and explode into even finer droplets. Finally electrostatic repulsion is sufficiently high to cause desorption of the analyte ions which are then passed to the Mass Analyzer for further detection.
-- MS instrument
¬†¬†¬†¬†We mainly apply HPLC (High Performance Liquid Chromatography) and 2-D LC (2-Dimension Liquid Chromatography) to separate and purify proteins from cells or tissues with the difference of protein polarity, hydrophobicity and affinity. HPLC includes Ion-exchange, hydrophobicity, and affinity chromatography. 2-D LC is composed of the successive application of Positive Ion-exchange and Reversed-Phase LC (or Negative-Ion exchange and Reversed-Phase LC column) to separated proteins mixture. Finally the target proteins are isolated from Reversed-Phase column and removed salt and impurity to decrease the loss of proteins in purification process.
¬†¬†¬†¬†This technology is used in the analysis of primary structure of protein. Two steps will be involved in the N,C-sequencing. The first step is to cut amino acid one by one from N- by chemical method; The second step is to separate protein by HPLC through separating and analyzing amino acid. N-sequencing uses Edman degradation method through ¶Ń-NH2 of proteins and peptides connecting with PITC and degrading , transforming to PTH-AA, and then the derivative is separated by C-18 RT- HPLC. The method of C-sequencing is similar to N-sequencing method, through ¶Ń-COOH of proteins and peptides activated and alkylated by reagents and then degraded to derivatives. Finally the derivatives are separated and analyzed by HPLC.