Structure Determination

Structural data is of high importance for peptide and protein characterization; however, data need to be obtained on highly purified samples. First of all, the primary structure can be determined by amino acid analysis, peptide mapping or Edman degradation, where the protein is degraded from the N-terminal and the sequence of amino acids determined subsequently. A similar procedure can be performed from the C-terminal. Protein modifications and posttranslational modifications such as glycosylations are thus identified. Furthermore, the primary structure along with effects from chemical degradation can also be determined by mass spectrometry (MS) (149,150).

The 3D structure of proteins, the secondary and tertiary structure, can be determined by Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and fluorescence. The maintenance of the globular structure is essential for the biological and pharmacological activity of proteins. The assessment of structure can be complex and often several complementing methods are applied, for example, X-ray crystallography, nuclear magnetic resonance (NMR) as well as FTIR, CD, and fluorescence spectroscopy. Many of these methods can also be used after preparation of the formulation to determine alterations caused by addition of excipients. FTIR is often applied when studying structural changes in proteins induced by various factors and when studying proteins in different environments and in different sample forms, for example, in solid or solution (151-153). It is a sensitive technique and is often used to monitor the a-helix-P-sheet refolding that follows aggregation (48). CD is also

Table 7 Useful Methods for Determination of Protein Structure and Stability as Well as Characterization of the Formulation

Technique

Information obtained

Remarks

Structure and stability

Amino acid analysis Edman degradation MS

Peptide mapping +

mass spectroscopy RP chromatography

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