**2.1.1 High Pressure Liquid Chromatography (HPLC)**

HPLC is a favorable method for peptides separation, because the HPLC can complete the separation in a short time under suitable chromatographic conditions, and more importantly, HPLC is capable of producing polypeptide of bioactivity at the preparative scale. Many scholars therefore have done substantive work in looking for the best conditions for separating and preparing polypeptide substances. How to maintain the activity of polypeptide, how to select stationary phase material and eluent type, how to make analytic determination are all contents of the present study. Common methods include: reversed phase high pressure liquid chromatography (RP-HPLC), hydrophobic interaction chromatography (HIC), Size-Exclusion chromatography (SEC), Ion-Exchange chromatography (IEC), Chromatography of Membrane Protein (CMP), High-Performance Displacement Chromatography (HPDC) and Perfusion Chromatography (PC).

#### **2.1.2 Affinity Chromatography**

Affinity Chromatography (AC) is the method of separating substances based on the specific affinity between ligand connecting to the stationary phase matrix and the ligand having interaction with the specificity. Since 1968 when Cuatrecasas put forward the concept of affinity chromatography, in searching for the specific affinity interaction substances many combinations have been found, like antigen-antibody, enzyme-substrate, agglutinin-polyose, oligonucleotides and their complementary strands. For the separation of polypeptide substances, currently the monoclonal antibody or biological simulation ligand can be used for affinity to such substances. These ligands can be natural or artificially synthesized according to their structure. Immobilized Metal Affinity Chromatography (IMAC) is an affinity method developed in recent years. Some metal ions are chelated on the stationary phase substrate, like Cu2+, Ni2+ and Fe3+. The magnetic beads can be chelated through the coordination bond to connect polypeptides that contain Lys, Met, Asp, Arg, Tyr, Glu and His on the side chain. In

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proteins/polypeptides); ion exchange magnetic beads include MB-WCX (purification and enrichment of acid proteins with the cation-exchange chromatography technology) and MB-WAX (purification and enrichment of basic proteins with the anion-exchange chromatography technology); glycoprotein magnetic beads include ConA and ConB (for purification and enrichment of glycoprotein); the immune fishing liquid chips include immune affinity magnetic bead ProteinG (the ProteinG on the magnetic bead can combine

Polypeptide separation technologies mentioned above are combined to use in practice. Different separation tools will be used according to the nature of polypeptide. In particular in the post genome era, researches on the proteome are deepened and people are making continuous progress in tools for separating polypeptides and proteins. They have comprehensively utilized natures of proteins and polypeptides and adopted both the routine protein and polypeptide extraction methods aforementioned and also the high efficiency liquid chromatography, capillary electrophoresis and 2-d electrophoresis, to get as

The Edman degradation method used for sequencing can obtain a precise peptide sequence, therefore making it a major method for protein identification. Yet its sequencing speed is slow and expansive. With technical breakthroughs in microsequencing and speed, Edman degradation method will exert a major role in proteome researches. The C-terminal Maxam-Gilbert method, similar to Edman, has been studied for years and produced automated

Amino acid composition analysis is frequently used for protein identification owing to its low cost. Different from the peptide mass or sequence tags, amino acid composition analysis identifies proteins by utilizing the specific amino acid component of different proteins. This method can be used for identifying 2-DE separated proteins. The radio-labeled amino acid is used to determine amino acid components of proteins, or the proteins are converted to the PVDF membrane and after the automatic derivation of amino acid, undergo chromatographic separation to obtain data. Then, inquiries are made to the database to rank proteins in the database by the amount of difference of two components, as a result, the top ranking proteins having greater reliability. Yet this method has some defects: slow speed and require a great amount of proteins or peptides; restricted in the ultramicro analysis;the possible amino acid variation due to the incomplete acidic hydrolysis or partial degradation.

Mass Spectrometry (MS): MS is an analytical technique that measures the mass-to-charge ratio (or mass) of charged particles, molecules or molecular fragments. MS provides information of molecular weight, molecular formula, isotopic element composition of

with any one antibody, for screening specific antigens).

many polypeptides as possible.

**2.2.2 Amino acid composition analysis** 

**2.2.3 Mass Spectrometry** 

**2.2 Sample detection** 

**2.1.6 Systematic applications of polypeptide separation engineering** 

**2.2.1 Detection of N-terminal sequence with Edman degradation method** 

analyzers, but its reaction efficiency is low and usually requires more samples.

particular, structures with the peptide sequences containing His-X-X-X-His are easiest combined to the metal ion affinity column, featuring good purification effect.
