E-Book Overview
Amino acid analysis is widely used in biotechnology, biomedical, and food analysis laboratories. Amino Acid Analysis Protocols constitutes a major collection of these indispensable analytical techniques, both classic and cutting-edge, of high utility for answering specific biological questions. Common methods include those based on HPLC or gas chromatography separation and analysis after precolumn derivatization. New techniques based on capillary electrophoresis separation, high-performance anion exchange chromatography, and mass spectrometry are also presented. Since results depend heavily on the quality of the sample, most contributors have devoted a section to sample preparation, particularly to the collection and storage of bodily fluids. A new method for desalting samples prior to hydrolysis is also provided. Each method is described in step-by-step detail to ensure successful experimental results, and contains helpful notes on pitfalls to avoid, and variations that enable the methods to be used with different systems. Up-to-date and highly practical, Amino Acid Analysis Protocols offers analytical and clinical chemists, as well as a broad range of biological and biomedical investigators, a rich compendium of laboratory tools for the productive analysis of both common and uncommon amino acids.
E-Book Content
Methods in Molecular Biology
TM
VOLUME 159
Amino Acid Analysis Protocols Edited by
Catherine Cooper Nicolle Packer Keith Williams
HUMANA PRESS
Amino Acid Analysis
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1 Amino Acid Analysis An Overview Margaret I. Tyler 1. Importance and Utility Amino acids are found either in the free state or as linear chains in peptides and proteins. There are 20 commonly occurring amino acids in proteins, which are shown in Table 1. Amino acid analysis has an important role in the study of the composition of proteins, foods, and feedstuffs. Free amino acids are also determined in biological material, such as plasma and urine, and in fruit juice and wine. When it is performed on a pure protein, amino acid analysis is capable of identifying the protein (2,3, and Chapter 8 in this volume), and the analysis is also used as a prerequisite for Edman degradation and mass spectrometry and to determine the most suitable enzymatic or chemical digestion method for further study of the protein. It is also a useful method for quantitating the amount of protein in a sample (see Chapter 2 in this volume) and can give more accurate results than colorimetric methods. 2. Historical View The earliest experiments on the acid hydrolysis of proteins were performed by Braconnot in 1820, in which concentrated sulphuric acid was used to hydrolyze gelatin, wool, and muscle fibers (4). Various reagents for performing protein hydrolysis were tried over the next 100 years, with 6 M HCl becoming the most widely accepted reagent. In 1972, Moore and Stein (5) were awarded the Nobel Prize for developing an automated instrument for separation of amino acids on an ion-exchange resin and quantitation of them using ninhydrin. More recently, high-performance liquid chromatographs (HPLCs) have been configured for amino acid analysis. Some methods use postcolumn derivFrom: Methods in Molecular Biology, vol. 159: Amino Acid Analysis Protocols Edited by: C. Cooper, N. Packer, and K. Williams © Humana Press Inc., Totowa, NJ
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Table 1 Common Amino Acids
3 letter
1 letter
Essential for humans (1)
Asp Glu
D E
No No
Ala Asn Cys Gln Gly Ile Leu Met Phe Ser Thr Trp Tyr Val
A N C Q G I L M F S T W Y V
No No No No No Yes Yes Yes Yes No Yes Yes No Yes
Arg His Lys
R H K
Yes Yes Yes
Pro
P
No
Symbol Name Acidic amino acid