Created: 1st December 2000, last updated: 30th December 2000, © 2000 ABRF
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. 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. It may be in these details that the book will serve the widest audience by suggesting specific reagents and protocols. Generally, more specialized topics have been emphasized in this volume; readers seeking background or introductory information will find copious references to earlier literature. Conversely, those concerned with problematic amino acid analyses, such as complex biologic samples or posttranslational modifications, will want to mine this volume for helpful insights and sample-handling tips.
Reviewed by Alan Mahrenholz