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Cells contain complex protein machinery that drives their repeated divisions, but they also have "checkpoint proteins" that prevent this from happening if conditions are not right. If their DNA is damaged, for example, the cell cycle is arrested. Now in a Perspective, Weinert synthesizes the results presented in three papers in this issue and describes a biochemical pathway that links for the first time the checkpoint proteins that sense DNA damage and the cyclin-dependent kinase-cyclin complex that is the "engine" that drives the cell cycle.

Although proteomics indicates a new view of mass protein analysis, it is in fact a synthesis of much that has been developing over the past twenty five years. In Chap. 2 through 7 of this book the status of the technology has been discussed, and how the different elements are integrated in proteome studies has been enunciated in Chap. 1. In Chap. 8 the emerging impact of proteomics in medicine has been considered. Here our brief is to take a broader, more biological view of where proteomics is (and will be) impacting on biology in general, particularly in areas of biotechnology that transcend medicine. It is important to realise that the field of proteomics is opening up as this book is being written. Thus much of the existing impact of proteomics involves use of the technology that is limited to 2-D gel mapping of complex protein mixtures. It is not surprising that to date there has been little characterisation of the proteins displayed, as only now are dedicated proteome facilities being established. However, current results do give a glimpse of the dramatic impact that proteomics is likely to have on all fields of biological sciences in the future.

The apparatus required for the regulation of gene expression varies widely in complexity. At one extreme, regulation can be achieved by one or two proteins acting at a DNA locus that encompasses 30–50 bp. However, such a simple picture is not usually (if ever) the whole story. Much more common are complex systems that typically involve several proteins acting over much larger segments of DNA. At these extended loci, one finds that function depends not only on a principal actor and a regulator (e.g., RNA polymerase and a transcriptional activator) but also on additional components. This chapter focuses on two of the best studied of such accessory factors: HU and IHF. We review the structure of these proteins, their influence on DNA conformation and topology, the regulation of their expression, their function in several well-studied systems, and their roles in homeostasis of E. coli. In order to focus on these questions, the present work has not presented all that is known about these proteins. More information can be found in several other surveys.1–6 For reviews of other accessory factors of E. coli—e.g., Fis, H-NS, Crp and Lrp—the reader is directed to chapters 12 and 20 of this volume and to other sources.7–11

The process of milk formulas for infants production is complex and multistage and includes the thermal processing. There is data, that different regimens of thermal processing influence on the structure of the protein-carbohydrate complexes, which can affect the formula’s protein uptake. It was shown, that interaction between complex protein units of the formula and the cells of the Peyer’s patches increased the risk of allergic reactions development in infants with predisposition to atopy. The influence of denaturation of milk formulas on the tolerance of them is discussed in this article.

In this paper we describe a specific staining method for γ-carboxyglutamic acid (Gla)-containing proteins in polyacrylamide gels. The procedure is based on the colorimetric detection of Gla using 4-diazobenzenesulfonic acid and has the advantage of being simple and fast (1 h). The detection limit is 9.4 pmol for prothrombin and 150 pmol for osteocalcin. It is demonstrated that Gla-proteins can be visualized independent of the presence of an excess of contaminating non-Gla-proteins. The technique may be used for screening of large number of fractions during the purification of Gla-proteins from complex protein mixtures.

The standard electrofractionation technique simulated the liquid chromatography to create a continuous elution system. Though this continuous lower buffer flow electrofractionation system served as an excellent model for preparative electrophoresis, the restricted current flow caused by electric resistance arising from the column holder prevented the application to the purification of complex protein mixtures. To overcome the electric current problem, the stopped lower buffer flow electrofractionation (SFE) system is designed to work without using a column holder and use prestained markers as the detection tool of protein position during the electrofractionation process. Through this improvement, the SFE system widens the availability of electrofractionation to the purification of complex and low-concentration protein mixtures. In this report, the efficiency of this technique is shown through the partial purification of a 32P-labeled protein in the rat liver microsome.

Purpose: Conventional diagnostic techniques for detecting Mycobacterium avium infection are far from satisfactory. As serodiagnostic tests for M. avium infection have been shown to be simple and rapid, the present study was carried out to identify and evaluate M. avium secretory protein(s) of diagnostic potential. Materials and Methods: Initially, by differential immunoblotting, a specific protein band of 45-50 kDa was recognized. Anion exchange column chromatography was used for purification of proteins. After fractionation, blast search was carried out. Further immunoreactivity studies were done with M. avium and Mycobacterium tuberculosis infected mice sera. Clinical utilization was confirmed by conducting indirect enzyme-linked immunosorbent assay (ELISA) with serum samples from mycobacterial infected patients. Results: A complex of three proteins (Aro-Tal-AST) of molecular weight ~48 kDa, shown to be Aro A homologue (Aro), transaldolase (Tal) and aspartate transaminase (AST) by blast search was separated. Immunoreactivity studies of purified complex protein with mice sera confirmed it to be specific for M. avium infection. Indirect ELISA with patient samples further confirmed it to be M. avium infection specific. Conclusion: Aro-Tal-AST protein is specifically recognized by patients infected with M. avium and can be used as a marker for simple and rapid ELISA based tests for differential diagnosis of M. avium infection in patients with M. avium complex (MAC).

Automated chromatography techniques such as high-performance (high-pressure) liquid chromatography (HPLC) or fast protein liquid chromatography (FPLC) represent a promising, new approach toward resolving the complex protein mixture of pancreatic juice. To achieve highly reproducible and rapid resolution of pancreatic protein profiles, reversed-phase, hydrophobic interaction, and ion-exchange techniques are employed. Until now, none of the respectively reported methods has provided a convenient, specific procedure for diagnostic purposes. However, experimental data prove that these methods are very sensitive and furthermore, easy to handle. Therefore, it seems possible to utilize such methods for diagnostics in future.

Publisher Summary This chapter discusses the feasibility of isolating peptides from a model peptide mixture and from a complex protein digest in single micropreparative separations. The peptides are recovered in sufficient purity and yield for characterization by the standard protein sequencing methodology. Analytical peptide maps of proteins have been well documented for both narrow-bore RP-HPLC and free solution capillary electrophoresis (CE). The quantities required for these analyses are impressively small, that is, in the low to sub-picomole range. However, the utilization of these techniques in a preparatory application is dependant on both the recovery of the peptides and on the sensitivity of the characterization method (protein sequencing). Adequate sequence information from narrow-bore RP-HPLC separations requires the loading of at least 50 pmol of digested protein. The recovery levels of peptides from micropreparative CE at load levels of approximately 100–150 pmol are encouraging. Fractions obtained from micropreparative separations appear to contain relatively low levels of background contamination.

We describe two general approaches to the utilization of DNA origami structures for the assembly of materials. In one approach, DNA origami is used as a prefabricated template for subsequent assembly of materials. In the other, materials are assembled simultaneously with the DNA origami, i.e. the DNA origami technique is used to drive the assembly of materials. Fabrication of complex protein structures is demonstrated by these two approaches. The latter approach has the potential to be extended to the assembly of multiple materials with single attachment chemistry.

To the Editor: The erythrocyte skeleton is a complex protein network responsible for the shape and the physical properties of red blood cells (RBCs), such as deformability and resistance to mechanical stress. Spectrin is the major constituent of this membrane skeleton and represents about 25% in

AGGREGATES OF DIFFERENT types of amyloid-forming peptides have different chiralities, a new study shows ( J. Am. Chem. Soc. , DOI: 10.1021/ja800328y). The finding gives clues about how these different peptides assemble and raises questions about the relationship between amino acid sequence, handedness, and amyloid-related ailments such as Alzheimer’s disease. Amyloids are complex protein aggregates with multiple levels of organization. Noa Rubin, Emanuel Perugia, Michal Goldschmidt, Mati Fridkin, and Lia Addadi of the Weizmann Institute of Science in Rehovot, Israel, have now used scanning electron microscopy (SEM) to learn more about one aspect of this hierarchy—amyloid chirality. The researchers examined three different amyloid fibers, each made from multiple copies of a disease-associated peptide segment from a different protein. They found that fibers made from a serum amyloid A peptide (SAA) possess a right-handed twist, and fibers from the other two peptides twist leftward. Amyloid fibers from diffe...

Protein phosphorylation is one of the most biologically relevant and ubiquitous post-translational modifications. The analysis of protein phosphorylation is very challenging due to its highly dynamic nature and low stoichiometry. In this article, recent techniques developed for phosphoproteome analysis are reviewed with an emphasis on the new developments in this field in China. To improve the performance of phosphoproteome analysis, many novel methods, either by application of new separation mechanisms or by adoption of new separation materials, were developed to specifically enrich phosphopeptides from complex protein digests. A series of new materials, including nanostructure materials, magnetic materials, and monolithic materials, were applied to prepare immobilized affinity chromatography or metal oxide affinity chromatography to improve the performance of phosphopeptide enrichment. Besides, new software tools were also developed to validate phosphopeptide identification and predict kinase specific phosphorylation sites.

INTRODUCTIONThis protocol describes the analysis of a proteome using multidimensional protein identification technology (MuDPIT), which couples 2D-LC to MS/MS, to resolve and identify peptides from complex mixtures. In this method, a pulled capillary microcolumn is packed with two independent chromatography phases: a strong cation exchanger and reversed-phase matrix material. Once a complex peptide mixture is loaded onto the system, no additional sample handling is required, because, as the peptides elute from the column, they are directed into the ESI ion-trap mass spectrometer, where they are ionized, mass-selected, and fragmented. Finally, advanced search algorithms match the fragmented peptides to their respective proteins in a database.

INTRODUCTIONProteins associated with insoluble particulate matter from a whole-cell lysate, or carefully prepared membrane samples, can be analyzed using MuDPIT analysis. However, the preliminary steps provided in this protocol are required to prepare a usable complex protein mixture from these types of samples.

INTRODUCTIONNanoliter-LC coupled to tandem mass spectrometry (nano-LC-MS/MS) permits the rapid and sensitive determination of protein-protein interactions. By using a specific purification technique such as coimmunoprecipitation or affinity purification in conjunction with nano-LC-MS/MS, not only are proteins identified, but specific protein-protein interactions are elucidated as well.

Successful long-term storage of a "smart dust" device integrating biomolecular motors and complex protein assemblies has been demonstrated using freezing or lyophilization, which implies that fabrication and application can be separated even for complex bionanodevices.

Many neurodegenerative diseases share a common feature: aggregation and deposition of abnormal proteins. The cellular mechanisms that cause these diseases can be studied by animal models. A novel approach to determine changes in protein expression is proteomics, i.e. separation of complex protein mixtures by two-dimensional-electrophoresis and subsequent identification of the proteins by mass spectrometry. In this contribution Huntington's and Parkinson's disease have been studied in two distinct mouse model systems using this approach. We identified disease-related proteins further upstream or downstream of the main disease pathway and proteins changed as a consequence of disease. Moreover, we found up-/down-regulated proteins which both diseases have in common. By comparing these results with protein changes associated with ageing in mice we identified shared protein changes as well. These data indicate that neurodegenerative diseases and ageing possibly affect the same cellular pathways.

As display manufacturers continue to develop increasingly thinner and flatter screens, one start-up company in the US is bucking the trend. Actuality Systems, based in Massachusetts, has recently launched a full-colour "volumetric" display that brings 3D images to life. The display, which was unveiled at the Society for Information Display's International Symposium in Boston in the US at the end of May, could help air-traffic controllers to visualize the sky surrounding a busy airport or drug developers to understand complex protein structures. It could even become the ultimate computer-games companion.

Phospholipase A2 (PLA2) activity was measured and partially characterized in prostasomes isolated from human seminal plasma. The results show high PLA2 activity in seminal plasma with a threefold enrichment in the isolated prostasomes. Highest activity was found at pH 8.0 and 10.5, and there was an absolute requirement for calcium with almost total inhibition of PLA2 activity in the presence of 1 mM EDTA. Analysis with two-dimensional gel electrophoresis of prostasome material revealed a complex protein pattern with most of the proteins in the molecular weight interval of 10,000–90,000 daltons. The possible role of PLA2 in prostasomes is discussed.

Combination of double-labeling and two-dimensional polyacrylamide gel electrophoresis allows high precision comparison of proteins from two different cultured cell lines. Extracts of cell lines labeled with 14 C and 3 H, respectively, are mixed and analyzed on a single two-dimensional gel. Fluorography detects both isotopes, but autoradiography through carbon paper detects only 14 C. Proteins can be classified into classes common to both cell lines and those specific to each cell line. The method should be useful for a wide range of biochemical studies.

High-resolution two-dimensional electrophoresis (2-DE) has reached a technological level that allows us to resolve most of the numerous unknown protein species of a mammalian organism if appropriate strategies are used. We will discuss the problems of classification and characterization of proteins and propose a systematic approach to the analysis of the total protein complex. Both a comprehensive as well as a pragmatic approach towards systematic analysis have been considered. A "complex protein database" is suggested and considered with regard to various uses. A systematic analysis of the mouse proteins has been started and some of the preliminary results are summarized here. In particular, genetic properties of the proteins were investigated and are presented in order to demonstrate the significance of a systematic analysis of proteins for research and practical application (e.g. mutagenicity testing). A concept is presented for sequencing the coding DNA of mouse and man, starting with a systematic analysis of mouse proteins and then using two recently developed methods - microsequencing of proteins from spots of 2-DE protein patterns, and utilization of the relatively short N-terminal sequences obtained - to produce the corresponding cDNA's of these proteins.

An improved high-resolution two-dimensional gel system for separating complex protein mixtures is described that allows a threefold increase in the number of proteins detected. Like the original O'Farrell system, proteins are separated in the first dimension by isoelectric point and in the second dimension by size. The improved resolution results primarily from a 2.5-fold increase in the size of both dimensions. Although best resolution is obtained by application of 5 × 106 cpm, as much as 150 μg of protein may be applied without appreciable loss of resolution. Useful separations may be made with up to 1.5 mg. By doubling the thickness of both dimensions, as much as 3 mg of protein can be separated into 300–400 separate peaks.

A complex protein fraction of mouse brain was subjected to dye ligand chromatography with various dye ligands. The proteins that were bound by the dye—gel matrix and also the non-binding proteins were separated by high-resolution two-dimensional electrophoresis. The protein patterns obtained were compared. The results show that a large number of different protein species bind to dye ligands and do not occur in the eluate. Red A was the most efficient dye in isolating an individual protein class from a complex tissue extract. Moreover, we found that many of the binding proteins did not cross-react among different types of dye ligands. Orange A and Blue B were the most unrelated dyes among those compared. Our investigation shows that dye ligand chromatography can be used as a means (among others employed previously) of fractionating and classifying the enormous number of different protein species in a mammalian tissue when combined with high-resolution two-dimensional electrophoresis.

Polyacrylamide films containing different glycosaminoglycans have been applied to the study of the Alcian Blue and combined Alcian Blue--Safranin O staining procedures. It was found that the polyacrylamide matrix can be interpreted as some kind of 'barrier' around the substrate molecules, a situation which can be compared to a certain extent with what occurs in situ, where complex protein molecules can likewise form a 'barrier'. The Alcian Blue staining of the model films was found to follow the Lambert-Beer law. The time to reach optimal dye binding depended on the concentration of the glycosaminoglycan enclosed in the model films and on the concentration of Alcian Blue in the dye solution. Lowering the pH of the dye solution appeared to increase the rate of staining. Optimal staining of model films in the presence of salt or urea was not possible, because under these conditions the pores of the polyacrylamide matrix became blocked. Alcian Blue was found to bind irreversibly to the glycosaminoglycan molecules enclosed in the polyacrylamide films. The results of the combined Alcian Blue-Safranin O staining applied to model films appeared to be highly dependent on the amount of Alcian Blue bound to the glycosaminoglycan in the first step of the double staining procedure. No specific differences were noticed between the behaviour of the different glycosaminoglycan-Alcian Blue complexes towards the Safranin O binding in the next step. As the theoretical basis for the application of the combined Alcian Blue-Safranin O staining was also found not to be completely valid, the conclusion was reached that this double staining cannot be used for the histochemical identification of glycosaminoglycans. The colour retained by a certain glycosaminoglycan-containing part of the specimen only delivers information about the accesibility of that part for Alcian Blue.

A technique has been developed for quantitating specific proteins present in a complex mixture, using a two-stage electrophoresis procedure. It consists of homogenizing a relevant portion of an isoelectric focusing gel into a slot of sodium dodecyl sulfate-polyacrylamide slab gel. After electrophoresis, proteins form bands which can be stained and analyzed by standard densitometric procedures. Radioactively labeled marker protein is used to monitor recovery, so that accurate quantitative measurements can be obtained. This method has been used to measure the abundance of actin in Drosophila egg chambers. The technique is applicable to the quantitation of many specific proteins among complex protein populations.

Artemia spp. of various geographic origins, yearly collection lots, developmental stages, and a binary geographic mixture were analyzed by thin layer polyacrylamide gel isoelectric focusing (IEF). IEF of geographical Artemia samples yielded unique profiles for each population in the pH range employed. Parthenogenetic populations of Artemia exhibited IEF patterns which were, generally, dominated by fewer proteins; bisexual populations exhibited more evenly distributed, complex protein patterns. There were similarities reflected in IEF profiles of geographically isolated Artemia descended from a common ancestry. Yearly collections of Artemia exhibited no apparent differences in IEF protein patterns. There were differences noted in three developmental stages of Artemia . A binary mixture of geographic populations yielded IEF patterns intermediate to those of the component pure samples.

Binding of non-ionic detergents is a characteristic property of ‘intrinsic’, amphiphilic membrane proteins [ 1,2] and there is currently an increasing interest in methods which permit detection of such detergentbinding. In this paper it will be shown how incorporation of 14Cor 1251-labelled Triton X-100 in agarose gels renders detection of amphiphilic proteins possible in crossed immunoelectrophoresis of complex protein mixtures. Due to binding of Triton X-100, such proteins give rise to radioactive precipitates which may be selectively visualized by autoradiography.

Human heavy chain diseases offer an opportunity to examine the molecular events taking place in cells which produce naturally occurring structural variants of a well-studied complex protein. Extensive structural analyses have been carried out on many of these proteins and have revealed the existence of certain characteristic features. Current studies are beginning to elucidate the molecular biology underlying these structural features.

The applicability of the methodology of disc electrophoresis to the determination of the component molecular weights of complex protein mixtures has been investigated. To this end, variable factors in the formation of polyacrylamide gel columns have been studied, along with their influence on the determination of relative mobilities. The effect of gelling time was demonstrated as a major factor in this regard, and the influence of catalyst and monomer concentration on this parameter defined. Migration of proteins of established amino acid composition at different pH's has been shown to vary in a parallel fashion to the alteration in charge of the protein. As a result of these investigations, the adoption of a standardized set of conditions which allow for the accurate estimation of the molecular weights of the protein components of biological systems has been made possible, a method that seems capable of wide application in assessing protein interrelationships.

A detailed study of the chromatographic fractionation of baleen S-carboxymethyl-kerateines has been made, using a system of automated step-wise elution. A comparison of the total kerateine preparations from baleen cells of widely differing inorganic content suggests a variation in protein proportions with the degree of mineralisation. The type of chromatographic system used is described and its reliable reproducibility suggests it to be suitable for quantitative analyses of complex protein mixtures.

It has now become of interest to study the structure of the synthetic polypeptides as they form simple models to use in the study of the more complex protein structure. In a very wide field, this has been done from the point of view of the helical and extended forms of the α and β polypeptide structures, and many polypeptide configurations have been characterized. In this paper, studies are made of the α, β and ω forms of the poly-β-benzyl- l -aspartates and their structural transformations on heating, including the types of energy transforms (endothermic or exothermic) accompanying these modifications. These latter changes have also been followed by X-ray and i.r. studies which have enabled us to characterize the forms unequivocally. In one of the poly- β-benzyl- l -aspartates, the transformation on heating has been shown to be α → ω → β; α → ω transformation is exothermic and the ω → β endothermic in character.

Lp(a)-lipoprotein of human serum, when isolated by ultracentrifugation and gelfiltration on Sepharose 4 B, may disaggregate into several components either spontaneously or by mild treatment with detergents

Mathematical modeling has established its value for investigating the interplay of biochemical and mechanical mechanisms underlying actin-based motility. Because of the complex nature of actin dynamics and its regulation, many of these models are phenomenological or conceptual, providing a general understanding of the physics at play. But the wealth of carefully measured kinetic data on the interactions of many of the players in actin biochemistry cries out for the creation of more detailed and accurate models that could permit investigators to dissect interdependent roles of individual molecular components. Moreover, no human mind can assimilate all of the mechanisms underlying complex protein networks; so an additional benefit of a detailed kinetic model is that the numerous binding proteins, signaling mechanisms, and biochemical reactions can be computationally organized in a fully explicit, accessible, visualizable, and reusable structure. In this review, we will focus on how comprehensive and adaptable modeling allows investigators to explain experimental observations and develop testable hypotheses on the intracellular dynamics of the actin cytoskeleton.

Stress fibers and focal adhesions are complex protein arrays that produce, transmit and sense mechanical tension. Evidence accumulated over many years led to the conclusion that mechanical tension generated within stress fibers contributes to the assembly of both stress fibers themselves and their associated focal adhesions. However, several lines of evidence have recently been presented against this model. Here we discuss the evidence for and against the role of mechanical tension in driving the assembly of these structures. We also consider how their assembly is influenced by the rigidity of the substratum to which cells are adhering. Finally, we discuss the recently identified connections between stress fibers and the nucleus, and the roles that these may play, both in cell migration and regulating nuclear function.

Elford, Grabar and Fischer1 have reported that the “antibody activity” of antipneumococcal horse-serum is associated with the “larger complex protein in the serum.” Thus it was estimated by the method of optimal proportions that 75% of the antibody passed a 140mμ membrane, rather less than one percent passed an 80mμ membrane, while the filtrate from a 54mμ membrane showed no trace of antibody-activity.The membranes used in the present experiments were prepared by the method of Elford2 with certain modifications described by Bauer and Hughes.3 For filtration, Type I antipneumococcal horse-and rabbit-sera were diluted 1:5 in broth. Concentrated antipneumococcal horse-serum was diluted 1:10 in the same reagent. For evaluating the amount of antibody in the filtrate the quantitative precipitation method of Heidelberger, Sia, and Kendall4 was used. Because of the difficulties in securing large amounts of filtrates complete analyses over a wide range of amounts of polysaccharide were impossible. Therefore an arb...

About 10 years ago, O’Farrell combined two electrophoretic methods which separated proteins by different parameters to produce a greatly enhanced separation of proteins in serum. This idea was developed by Anderson and Anderson (1978a) at Argonne Laboratory, and they have called their methodology the Iso-Dalt system (Anderson et al., 1978a; Anderson et al., 1978b).