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BOOK REVIEWS
Amino Acid Analysis Protocols. Edited by Catherine Cooper,
Nicolle Packer, and Keith Williams. Methods in
Molecular Biology series number 159.Totowa, NJ: Humana
Press, September 2000. Hardcover, 280 pp, $84.50
US.
This volume is what the title states; it is intended
as a benchtop reference that provides recipes and
procedures for amino acid analysis. An introductory
chapter by Margaret Tyler provides guidance to the
topics found among the 17 other chapters of the
book. Of these, several focus on standard amino acid
analysis strategies commonly found in core facilities:
postcolumn ninhydrin and AQC (AccuTag). The former,
by Macchi and colleagues, is focused on modifications
that occur (at low levels) during protein
expression in a pharmaceutical setting. Here the ninhydrin
system is well suited to resolving minor components
(e.g., norleucine, hydroxylysine, amino sugars,
and carboxmethylCys) amidst the high levels
analyzed. One useful feature of this article is a clear
illustration of typical (Excel) spreadsheet calculations
employed to obtain protein amino acid compositions.
The article on AQC by S. A. Cohen is clear and helpful,
particularly in providing the buffer recipes for the
analysis using Waters’ proprietary column. Shindo and
colleagues describe an application of AQC analysis to
identify blotted protein using the ExPASy Web Site.
The authors correctly emphasize the need for good
hygiene in sample handling and provide diagrams for
an isolation box and their version of a hydrolysis
vessel. In several places, the reader is reminded that
desalting protein samples is important for accurate
compositional analysis. To address this issue, there is
a brief contribution from Zhang and Denslow that
details simple desalting protocols.
Phenylisothiocyanate (PITC) as detection reagent
is discussed in comparison with new reagents and in
the analysis of complex biologic matrices. Woo presents
a comparison between butyl-isothiocyanate–
(BITC-) and benzyl-isothiocyanate– (BZITC-) derivatized
amino acids and the familiar PITC approach.
Journal of Biomolecular Techniques
11:177–178 © 2000 ABRF
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The primary motive was to shorten analysis time and
increase convenience by finding a volatile reagent. In
contrast to PTC chromatography, both BITC and
BZITC compounds provide separation of cysteine
and cystine. Although BITC possessed the desired
volatility, resolution on-column suffered (i.e., benzylthiocarbamyl
[BTC]-Asn and BTC-Ser co-eluted)
and sample stability was only about 8 hours. The
BTC derivatives appeared to provide superior resolution
in the same system relative to PTC compounds,
but the volatility of the reagent was similar to PITC.
It would be surprising indeed if laboratories abandoned
the established PITC reagent in favor of a new
reagent to gain incremental advantages, particularly
because the authors admit that analysis of cysteine
and cystine is undependable without prior derivatization
of thiols to stabilize them. Stark and Johansson
discuss PITC analysis in the context of lipid-containing
samples.
Two chapters provide examples of electrochemical
detection. One of these, by Jandik and colleagues,
competently discusses integrated pulsed amperometric
detection (IPAD) coupled to ion-exchange chromatrography,
which is widely used for carbohydrate
analysis. The balance of the chapters discuss techniques
that diverge from the norm, either in instrumentation
or sample source. Instrumentation and detection
strategies include a quick, universal approach
using Marfey’s reagent, the amperometric methods
noted previously, capillary electrophoresis detection,
precolumn and postcolumn OPA, and elegant flame
photometry/gas chromatography techniques (used for
analysis of O-phospho-amino acids and thiol-amino
acids). Among this last group of chapters, analysis of
blood plasma, foods, and modified amino acids are
the predominent subjects. The final chapter on protein
glycation products introduces mass spectrometry
as the detection device and provides a concise introduction
to this side reaction.
Although the chapters present disparate approaches
to amino acid analysis, editorial consistency
is maintained through the use of extensive footnotes
that, in many cases, provide useful general information.
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 177