Peptide Analysis

Amino Acid Analysis

 

Introduction

In many cases an exact knowledge of protein quantities is required for further protein chemistry applications. Amino Acid Analysis is the suitable tool for precise determination of protein quantities, but also provides detailed information regarding the relative amino acid composition and free amino acids. The relative amino acid composition gives a characteristic profile for proteins, which is often sufficient for identification of a protein. It is often used as decision support for choice of proteases for protein fragmentation.

The procedure includes:

  • Hydrolysis
  • Separation, Detection and Quantification

Hydrolysis

Hydrolysis is typically achieved by acid conditions. A standard procedure is hydrolysis with 6 M hydrochloric acid (24 hours, 110°C). These standard procedure is a compromise between time requirement and temperature. Sensitive amino acids (especially tryptophane and cysteine) will be partially destroyed. Gas phase hydrolysis and addition of other acids (e.g. propionic acid, TFA, methansulfonic acid) can be used to shorten the hydrolysis time and improve the yield of sensitive amino acids.

Separation, detection and quantification

Hydrolysed samples(amino acids)are derivatized for sensitive detection,separated by HPLC. Whereas post-column derivatization was typical earlier, pre-column derivatization has gained importance and can be achieved by a broader range of derivatization reagents. The use of internal and external standards is crucial. A selection of well suited products is listed below(see Table 2).


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