Book Reviews

Protein Purification Protocols,Second Edition

Paul Cutler (Editor), Methods in Molecular BiologySeries 244, Humana Press, Totowa, New Jersey, 2003,512 pagesISBN: 1-58829–067-0

The book “Protein Purification Protocols” edited by PaulCutler is a valuable collection of methods for proteinseparation and purification that can answer the severalrequirements necessary to obtain purified proteins in anyparticular case. It is enriched by notes and practical sug-gestions, similar to a collection of recipes from a famouschef. Although all procedures are not really describedstep-by-step so that they can be used by inexperiencedresearchers, who in any case can find these elsewhere,the critical steps and coordinated actions are pointed outvery well and much meticulous advice is provided makingthis a very useful instrument for advanced work.

The book includes sections which deal with proceduresfor protein fractionation starting from tissue or bacterialprotein extraction to final protein purification, and eachchapter is subdivided into an introductory part, whereproblems are defined and theoretical aspects of proce-dures chosen to answer specific requirements, followedby a methodological one.

Answers to the main questions concerning the quantity ofprotein required to carry out chemical, physical, kineticanalysis, or mass spectrometry analysis are described inchapters 13, 14 , 36, 40–42 with attention to the source ofthe proteins. Chapters 3, 7 and 8 are dedicated to theextraction of plant proteins, including organelles such asmitochondria and chloroplasts, whilst in chapters 4 and 5procedures for manipulating the levels of the desiredmicrobial or fungal proteins by control of growth mediaare described. However, procedures for fractionating pro-teins from biological fluids such as plasma, spinal fluid,and urine are not described. Detailed methods of pro-tein extraction from animal tissues are described in chap-ter 2, while those of subcellular fractionation with atten-tion to plasma membrane separation are discussed inchapter 6. Since membrane proteins are difficult to frac-tionate owing to their hydrophobic surfaces and particularsolubilizing properties and post-translational modifica-tions such as glycosylation, procedures to extract andpurify them are extensively explained in chapters 15, 18and 29. Finally, excellent overviews of fast protein liquidchromatography and reverse-phase chromatographyare provided in chapters 26 and 28, as well as in chapter34, where alternative approaches by electroelution to

obtain an enriched solution of a purified protein frompolyacrylamide gels after 1- or 2-D electrophoresis aresuggested.

Problems concerning the levels of protein purity neces-sary for different applications with retention of biologicalactivity are discussed in chapter 1, suggesting differentmethods that are further emphasized in chapters 16, 19and 11, which discuss affinity chromatography, immu-noaffinity chromatography and changing buffer at the dif-ferent steps of protein purification, respectively. In chap-ter 9, methods to avoid proteolysis and to measure andmonitor levels of protease activities are available withdetailed explanation of experimental evidence of proteo-lytic degradation and strategies of its prevention. In addi-tion, in chapters 32 and 33 several aspects and schedulesare respectively considered for lyophilization and storageof pure proteins.

This book takes us on a veritable guided tour throughthe majority of procedures of protein purification, under-lining the baseline explanations of specific questionsand proposing the most economic solutions with anabundance of comments and practical applications. Itis a theoretical manual able to gain a wide consensusparticularly within expert researchers in the proteomicfield.

Cecilia Sarto,University Department of Laboratory Medicine,Desio Hospital,Desio (Milano), Italy

Protein Arrays, Biochips, and Proteomics

The Next Phase of Genomic Discovery

Edited by Joanna S. Albala and Ian Humphery-SmithMarcel Dekker, 2003, 409 pagesISBN: 0-8247-4312-1

Following the completion of the human genome se-quencing project, well-established DNA microarrays andsophisticated bioinformatics platforms allow scientiststo take a global view of biological systems. In today’sproteome era, the time is ready for protein microarraysto analyse protein expression and study the interactionsof protein functions from a global perspective. In con-trast to DNA chip technology used to determine mRNAexpression, protein microarray technology is a muchmore ambitious endeavour. Within the world of proteins,not only their abundance has to be determined, but alsoinsights into their functional states have to be gained.Appropriate high-throughput and sensitivity can be

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achieved with protein microarrays. However, the amountof work associated with such solutions is still tremen-dous.

The recently published book on “Protein Arrays, Biochips,and Proteomics – The Next Phase of Genomic Discov-ery”, edited by Joana Albala and Ian Humphery-Smith,provides an excellent overview of protein arrays and bio-chips. It highlights their application in proteomic researchand deals with selected proteomic issues. Ian Humphery-Smith introduces the topic by giving a broad overview ofprotein biochips and array-based proteomics. All relevantaspects of array-based proteomics are discussed. Themost critical issue of array-based proteomics is the avail-ability of appropriate content. The limited availability ofhighly-specific capture molecules is the main differencebetween protein and DNA microarray technology. It isimpossible to predict high-affinity capture molecules forproteins purely on the basis of their primary amino acidsequence due to their most diverse and individual tertiarymolecular structures and their manifold possibilities tointeract simultaneously, even with different binding part-ners. And just to make things even more complicated:proteins tend to appear in complexes. Steady or dynamicpost-translational modifications such as glycosylation orphosphorylation have an additional influence on theirfunction. All these aspects are thoroughly discussed andpromising potential solutions regarding the visionary fieldof array-based proteomics are suggested.

In the second chapter Roger Ekins and Frederick Chu elu-cidate the fundamental principles of miniaturised multi-plexed ligand-binding. Ekin’s “ambient analyte theory”states that small amounts of capture probes are the man-datory prerequisites for the detection of analyte concen-trations with the highest possible accuracy. In addition tothe miniaturised format, an important advantage of micro-array assays is the ability to quantify different analytes inparallel without a substantial change in the analyte con-centration of the sample. Apart from the basic theoreticalaspects of microarray technology, this chapter also sum-marise high-quality microarray data that were generatedby Ekins and his coworkers nearly ten years ago. In thethird chapter, Brian Haab gives an overview of practicalapproaches to protein microarrays and provides detailson the principle set-up of the dual-colour labelling pro-cedure of antibody-antigen microarrays. The functionalimmobilisation of capture molecules discussed in thischapter, is further detailed in chapter 4, written by SteffenNock and Peter Wagner, as well as in chapter 5, writtenby Erik Wischerhoff. The essential prerequisite for thegeneration of expedient capture molecules is the avail-ability of the corresponding target proteins. It becomesclear that the development of effective methods for the

cost-effective, fast high-throughput generation of proteintargets comes first on the cumbersome path to generateappropriate capture molecules. This issue is addressedby Stefan Schmidt in chapter 6 and by the group of JoanaAlbala in chapter 7. Different aspects of high-throughputprotein expression and of miniaturised protein produc-tion are thoroughly discussed. The authors describe ahigh-throughput system for the expression and purifica-tion of eukaryotic proteins to provide the resources forstructural studies and protein functional analysis. In chap-ter 8, Eric Fung and Enrique Dalmasso describe an alter-native approach to screen for biomarkers involving theSELDI (surface enhanced laser desorption/ionization)technology which uses mass spectrometry as the read-out system. Cell extracts that are derived from differentsources are incubated on different spots on the sameadsorptive surface chemistry. Unbound or weakly boundproteins are washed away, whereas the whole variety ofnonspecifically captured target proteins can be analysedby mass spectrometry.

From chapter 9 onwards, emerging proteomic technolo-gies are discussed. The series starts with Anita Sara andJohn Yates III discussing the power of shotgun proteom-ics and its applications in the study of the yeast proteome.The major problem for whole proteome analysis is its verydynamic nature and the tremendous dynamic range ofprotein concentrations in biological samples. One singlegenome can give rise to an enormous number of pro-teomes. One promising approach to analyse such com-plex samples with current technologies is the combina-tion of on-line high-resolution liquid chromatography andtandem mass spectrometry. Applying this method, eachcomponent can be identified against the background ofthousands of other proteins. Such an approach has thepotential to detect post-translational modifications aswell. In chapter 10, David Hill and colleagues discussprotein interaction networks using the Y2H system. Sucha “gene-based direction”, referred to by the authors as“reverse proteomics”, can be performed on a genome-wide scale and results in networks defining protein inter-action or interaction maps detailing cellular function. Theprotein fragment complementation strategy is presentedin chapter 11 by Ingrid Remy and Stephen Michnick. Thisis another promising technology that has proven suitablefor studying protein-protein interactions in living cells. Theauthors discuss how this technology can be used for thegenome-wide mapping of biochemical pathways. Chap-ter 12, written by Wuxian Shi and colleagues, describesthe critical steps for high-throughput structural biologyapproaches and the impact structural genomics willhave on the fields of cell biology and biochemistry. Chap-ter 13, written by Shaw and colleagues, clearly demon-strates that the tremendous amount of data sets gener-

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ated within the field of microarray technology or proteom-ics are of no use if bioinformatic tools are not applied. Toanalyse the incredible amount of data, new data analysisplatforms are needed which in turn will lead to new para-digms or mathematical models. The book closes with anexpert insight in the dynamic market of protein microar-rays provided by Steven Bodovitz .

In summary, this book is an invaluable resource for anyresearcher of the academic or private sector looking intothe potential of protein microarray technology for prote-omic research. The very different aspects of this emergingfield are presented from different perspectives and pro-vides the readers with valuable information and knowl-edge.

Thomas Joos,NMI Natural and Medical Sciences Institute,University of Tübingen, Germany

Biomedical Applications of Proteomics

J.-C. Sanchez, G. L. Corthals, D. F. Hochstrasser (Eds.)Wiley-VCH, Weinheim, 2004, 435 pagesISBN: 3-527-30807-5

The book entitled “Biomedical Applications of Proteom-ics” is the first book summarizing recent developmentsin the field of biomedical proteomics research. It providesa picture of proteomics studies of the mechanisms ofvarious diseases with a common aim – to help answerquestions of diagnosis, prognosis, and therapy. Thebook is divided into seven main parts: Part I, Aspects inBiomedical Research; Part II, Blood Vessels; Part III, Can-cer; Part IV, Pharmaco-toxicology; Part V, Infectious Dis-eases; Part VI, Central Nervous System; Part VII, MassSpectrometry and Bioinformatics.

The first part introduces the aspects of biomedical re-search with a special focus on human material. It isexpected that the role of proteomics studies in clinicalmedicine will increase over the next years and medicinewill shift from generalized knowledge to personalizedpractice. However, ethical questions and patient consentneed to be accurately addressed. Most of the chapters inthe following parts first highlight the physiopathology ofdisease and then the proteomic findings are summarized.

Part II describes blood vessel related diseases includingatherosclerosis and stroke and demonstrates that up-dated proteomic procedures have to be applied and com-bined to monitor disease progression and to select ap-propriate biomarkers. Due to the complex compositionof atherosclerotic plaques, microdissection appears to

be important for evaluation of the various cell types con-tributing to the lesion. In the case of multisystemic andmultifactorial diseases such as neurodegenerative dis-eases, the simultaneous assessment by a panel of bio-markers appears to be important for characterization ofthe mechanisms of neurotoxic and thrombotic coupling.The SELDI-TOF technology could be successfully appliedin searching the proteins or peptides important for diag-nosis. Furthermore, the area of ligand-based vasculartargeting is promising for development of an anti-angio-genic treatment for cancer.

In part III the authors describe the biomedical applica-tions of proteomics in cancer. Cancer belongs to themost documented area using this technology and well-recognized authors contributed to each chapter. Thereader will find the proteomic investigation of renal can-cer, colorectal cancer, and ovarian cancer. The specificrole for HSP27 is summarized with respect to the differentpost-translational modifications in various cancers. Theinvestigation of lymphoblastoid and lymphoma cellstreated with a demethylating drug 5-azacytidine or withgalectin1 was selected to perform proteomic studies ofmalignant hemopathies. A specific chapter deals withprotein profiling of hematopoietic stem cells. In regard tothe fact that the therapeutic potential of stem cells ishuge, the extension of current knowledge on stem cellbiology is necessary and more comprehensive studieshave to be performed including stem cell proteome andits variations.

Part IV on pharmacology and toxicology provides exam-ples of the studies of mechanisms underlying cancerchemoresistance. The results obtained by using severalcell lines allowed possible mechanisms of resistance tobe suggested, however, the authors stressed that “thisis only the first step to analyze global protein expres-sion and to find candidate proteins that may be involvedin certain pathways . . .”. Another chapter in this partdescribes complex molecular mechanisms of diabetesmellitus. A series of proteomic studies have been per-formed in T1 DM and the results are expected to providethe molecular signature of the beta-cell destruction whichwill be helpful in increasing resistance of these cells toimmune mediators and associated increased survivaltime of transplanted islets.

In part V the proteome approach to infectious diseases,including infections caused by viral as well as bacterialrepresentatives, is presented. The first chapter of thispart presents data related to the host response by moni-toring acute phase and humoral response and influenceof host genetic background on the outcome of infectiousdisease. In the next chapter the authors describe theirstudies on human lymphocytes collected from healthy

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