**2.2.5 Others**

374 Biomarker

molecules and molecular structure of samples analyzed. It has been widely applied in protein and polypeptide analysis. In particular, it is suitable for the analysis and identification of polypeptide substances in the online analysis after separation and purification due to its hypersensitivity and rapidity. Commonly, MS includes electrospray MS (in the spray process, the continuous ionization method makes the polypeptide samples ionized), fast atom bombardment MS (FAB MS) and isotopic element MS. Among them, the Continuous-Flow Fast Atom Bombardment, cf-FAB) and the Electrospray Ionization (ESI)

Continuous-Flow Fast Atom Bombardment(cf-FAB): a kind of weak ionization technology, it is capable of ionizing peptides or small-molecular-weight proteins into the form of MH+ or (M-H). It is mostly applied in the separation and detection of peptides and has moderate resolution, with the accuracy greater than +0.2amu and flow rate of 0.5-1.5μl·Ml-1. In the determination, the mobile phase shall be added with 0.5%-10% substrate like glycerol and high organic solvents, so that samples can be sensitized at the detection probe. The cf-FAB is usually used together with HPLC and CEZ, to realize the purpose of isolation analysis. The

Electrospray Ionization (ESI): able to generate multivalent ionized proteins or polypeptides, allowing analyzing of proteins with the molecular weight reaching 100kD; its resolution is 1500-2000 amu and accuracy about 0.01 %. ESI is more suitable for the online analysis of proteins with large molecular weight and requires gasification or organic solvents for the sample sensitization. It has been a success to combine ESI and HPLC for separation and

MALDI-TOF MS: in this method, the ionization of polypeptide samples is realized with the substrate absorbing the laser energy. It is a tool for accurately determining the molecular mass in the current protein identification and particularly suitable for the determination of the molecular weight of mixed proteins and polypeptides, featuring high sensitivity and resolution. For the moment, it is a necessary tool for the proteomics research. Working with the coupling technique of the liquid chromatography, this method can identify polypeptides at a high efficiency. Especially, when MS technologies of different principles are coupled, they can not only obtain the molecular weight of polypeptides, but also determine the sequential

Nuclear Magnetic Resonance (NMR): NMR profiling has purely digital signals, excessive overlapping range (due to the large molecular weight) and weak signals, so it is seldom used in analysis of proteins and polypeptides. In company with the application of 2D, 3D and 4D NMR and the progress of molecular biology and computer processing technology, NMR has gradually become a main approach for analysis of proteins and polypeptides. NMR can be used for determining amino acid sequence and the content of components in mixtures. Yet some problems need solving if this method is used for protein analysis, for instance, how to give proteins with large molecular weight a specific shape to facilitate quantitative and qualitative analysis and how to reduce the data processing time, which are being studied by many scholars. Despite of its infrequent use in the protein analysis, NMR is extremely useful in analyzing small peptides with the molecules having less than 30 amino acids, in which case,

cf-FAB analysis methods have been built for many polypeptides and well applied.

structure. This technology will exert a decisive effect in the future proteomics study.

it can overcome the foregoing defects and realize rapid and accurate analysis.

analysis of GH and hemoglobin. ESI can also be used together with CEZ.

have just been developed in recent years.

**2.2.4 Nuclear Magnetic Resonance** 

Besides the foregoing methods, amino acid composition analysis, amino acid sequence analysis, Field Desorption Mass Spectrometer (FDMS), IR, UV spectra, circular dichroism spectrum, bioassay technique, tagging method and immunologic method have also been used for the result identification, analysis and detection of polypeptides.
