Analysing proteomic data

The rapid growth of proteomics has been made possible by the development of reproducible 2D gels and biological mass spectrometry. However, despite technical improvements 2D gels are still less than perfectly reproducible and gels have to be aligned so spots for identical proteins appear in the same place. Gels can be warped by a variety of techniques to make them concordant. When gels are manipulated to improve registration, information is lost, so direct methods for gel registration which make use of all available data for spot matching are preferable to indirect ones. In order to identify proteins from gel spots a property or combination of properties that are unique to that protein are required. These can then be used to search databases for possible matches. Molecular mass, pI, amino acid composition and short sequence tags can all be used in database searches. Currently the method of choice for protein identification is mass spectrometry. Proteins are eluted from the gels and cleaved with specific endoproteases to produce a series of peptides of different molecular mass. In peptide mass fingerprinting, the peptide profile of the unknown protein is compared with theoretical peptide libraries generated from sequences in the different databases. Tandem mass spectroscopy (MS/MS) generates short amino acid sequence tags for the individual peptides. These partial sequences combined with the original peptide masses are then used for database searching, greatly improving specificity. Increasingly protein identification from MS/MS data is being fully or partially automated. When working with organisms, which do not have sequenced genomes (the case with most helminths), protein identification by database searching becomes problematical. A number of approaches to cross species protein identification have been suggested, but if the organism being studied is only distantly related to any organism with a sequenced genome then the likelihood of protein identification remains small. The dynamic nature of the proteome means that there really is no such thing as a single representative proteome and a complete set of metadata (data about the data) is going to be required if the full potential of database mining is to be realised in the future.     

Mouse models for neurodegenerative diseases and a theory of proteomics

Proteome analysis is usually performed by separating complex cellular protein extracts by two‐dimensional‐electrophoresis followed by protein identification using mass spectrometry. In this way proteins are compared from normal and diseased tissue in order to detect disease related protein changes. In a strict sense, however, this procedure cannot be called proteome analysis: the tools of proteomics are used just to detect some interesting proteins which are then investigated by protein chemistry as usual. Real proteome research would be studying the cellular proteome as a whole, its composition, organization and its kind of action. At present however, we have no idea how a proteome works as a whole; we have not even a theory about that. If we would know how the proteome of a cell type is arranged, we probably would alter our strategy to detect and analyze disease‐related proteins. I will present a theory of proteomics and show some results from our laboratory which support this theory. The results come from investigations of the mouse brain proteome and include mouse models for neurodegenerative diseases.     

Gene therapy approaches for Parkinson's disease

Proteome analysis is usually performed by separating complex cellular protein extracts by two-dimensional-electrophoresis followed by protein identification using mass spectrometry. In this way proteins are compared from normal and diseased tissue in order to detect disease related protein changes. In a strict sense, however, this procedure cannot be called proteome analysis: the tools of proteomics are used just to detect some interesting proteins which are then investigated by protein chemistry as usual. Real proteome research would be studying the cellular proteome as a whole, its composition, organization and its kind of action. At present however, we have no idea how a proteome works as a whole; we have not even a theory about that. If we would know how the proteome of a cell type is arranged, we probably would alter our strategy to detect and analyze disease-related proteins. I will present a theory of proteomics and show some results from our laboratory which support this theory. The results come from investigations of the mouse brain proteome and include mouse models for neurodegenerative diseases.     

Proteomic approach to identify novel mitochondrial proteins in Arabidopsis.

An Arabidopsis mitochondrial proteome project was started for a comprehensive investigation of mitochondrial functions in plants. Mitochondria were prepared from Arabidopsis stems and leaves or from Arabidopsis suspension cell cultures, and the purity of the generated fractions was tested by the resolution of organellar protein complexes applying two-dimensional blue-native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Arabidopsis mitochondrial proteome was analyzed by two-dimensional isoelectric focusing/ Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 650 different proteins in a pI range of pH 3 to 10 were separated on single gels. Solubilization conditions, pH gradients for isoelectric focusing, and gel staining procedures were varied, and the number of separable proteins increased to about 800. Fifty-two protein spots were identified by immunoblotting, direct protein sequencing, and mass spectrometry. The characterized proteins cooperate in various processes, such as respiration, citric acid cycle, amino acid and nucleotide metabolism, protection against O(2), mitochondrial assembly, molecular transport, and protein biosynthesis. More than 20% of the identified proteins were not described previously for plant mitochondria, indicating novel mitochondrial functions. The map of the Arabidopsis mitochondrial proteome should be useful for the analysis of knockout mutants concerning nuclear-encoded mitochondrial genes. Considerations of the total complexity of the Arabidopsis mitochondrial proteome are discussed. The data from this investigation will be made available at http://www.gartenbau.uni-hannover.de/genetik/AMPP.     

Biochemical, biophysical, and biological properties of densonucleosis virus. I. Structural proteins.

The polypeptide composition of highly purified densonucleosis virus was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The viral proteins showed a different behavior in sodium dodecyl sulfate-gels in comparison with the marker proteins. Therefore, the molecular weights were estimated by analyzing the retardation of the electrophoretic mobility of these proteins in gels with increasing polyacrylamide concentrations. Four structural proteins with molecular weights of 49,000, 58,500, 69,000, and 98,000 were found, ant they were designated p49, p59, p69, and p98, respectively. There are several indications that p98 is a dimer of p49. The relative quantity of the structural proteins in a virion suggests that at least p49 (accounting for +/-70% of total protein mass) is a capsid protein and that there will be 12 capsomers per virion.     

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.     

The molecular scanner: concept and developments

Approaches aimed at deciphering the proteome have illustrated the need for relatively complex and highly sensitive methodologies. The major elements of proteome analysis, such as powerful protein separation and enzymatic processing, mass spectrometry and dedicated bioinformatics have been assembled in the development of the molecular scanner. This highly flexible and data-rich approach has combined the power of electrophoretic protein separation, the simultaneous digestion and transfer of proteins through an enzymatic membrane, the immediate use of the MALDI mass spectrometer to scan a collecting membrane, and the development of dedicated bioinformatics tools to perform protein identification and molecular imaging of the proteome. Clinical applications of the molecular scanner have also started to be developed for disease diagnosis in biological material.     

Interaction Proteomics

The term proteome is traditionally associated with the identification of a large number of proteins within complex mixtures originating from a given organelle, cell or even organism. Current proteome investigations are basically focused on two major areas, expression proteomics and functional proteomics. Both approaches rely on the fractionation of protein mixtures essentially by two-dimensional polyacrylamide gel electrophoresis (2D-gel) and the identification of individual protein bands by mass spectrometric techniques (2D-MS). Functional proteomics approaches are basically addressing two main targets, the elucidation of the biological function of unknown proteins and the definition of cellular mechanisms at the molecular level. In the cell many processes are governed not only by the relative abundance of proteins but also by rapid and transient regulation of activity, association and localization of proteins and protein complexes. The association of an unknown protein with partners belonging to a specific protein complex involved in a particular process would then be strongly suggestive of its biological function. The identification of interacting proteins in stable complexes in a cellular system is essentially achieved by affinity-based procedures. Different strategies relying on this simple concept have been developed and a brief overview of the main approaches presently used in functional proteomics studies is described.     

Purification and Characterization of a Salt-extractable Hydroxyproline-rich Glycoprotein from Aerated Carrot Discs

The salt-extractable hydroxyproline-rich glycoprotein (HRGP) of the cell wall of aerated carrot root discs has been studied by polyacrylamide gel electrophoresis. The predominant proline-labeled protein extractable from the cell wall is rich in hydroxyproline as shown by its specific loss of ³H from proline labeled in position 4 and its shift in electrophoretic mobility after labeling in the presence of an inhibitor of hydroxyproline synthesis. Unlabeled HRGP can be identified by staining gels for carbohydrate. The HRGP has been purified by ion exchange chromatography and CsCl gradient centrifugation. The HRGP consists of about 50% protein and 50% carbohydrate with an overall molecular weight of 86,000. The amino acid composition of the protein portion consists of 50% hydroxyproline, 19% basic amino acids, 12% serine, and 10% tyrosine. This glycoprotein accumulates in a salt-extractable pool in the cell wall beginning between 10 and 20 hours of aeration and may also become incorporated into the nonextractable portion of the cell wall.     

Proteins Specified by Herpes Simplex Virus XII. The Virion Polypeptides of Type 1 Strains

The polypeptides from purified virions of a herpes simplex 1 (human herpes-virus 1) strain, F1, which had been passaged a limited number of times in cell culture after isolation, formed 33 bands on electrophoretic separation in polyacrylamide gels cross-linked with N, N'-diallyltartardiamide in contrast to a maximum resolution of only 24 to 25 bands in gels cross-linked with N, N'-methylenebisacrylamide. This increase in the number of bands was due chiefly to an improved separation of glycosylated polypeptides from nonglycosylated polypeptides with which they co-electrophoresed on methylenebisacrylamide cross-linked gels. Purified virions of HSV-1 [F1] had a protein/DNA mass ratio of 10.7 +/- 0.96, and based on a DNA molecular mass of 85 x 10(6) to 100 x 10(6) the estimated weight of virion polypeptides ranges from 16.4 to 19.4 x 10(-16) g. The number of molecules of each polypeptide per virion ranged from less than 50 to 1,500. Comparison of the virion polypeptides of two HSV-1 strains with similar isolation and limited passage history with those of four HSV-1 strains with histories of numerous passages outside the human host showed a number of nonrandom variations in virion polypeptides. Thus, although the virion polypeptides of two strains with similar isolation and limited passage history could not be differentiated, strains with extended passage histories differed markedly from each other and from the limited passage strains in the number and electrophoretic mobility of noncapsid polypeptides and notably in those of the envelope.     

Structural rearrangements in active and inactive forms of hydrogenase from Thiocapsa roseopersicina.

The electrophoretic behavior of Thiocapsa roseopersicina hydrogenase on sodium dodecyl sulfate gels demonstrates that the protein exists in two active forms, A1 and A2, which may be interconverted. Each of these forms has a characteristic electrophoretic mobility and differs in its sensitivity to O2. Form A1 is O2-labile and converts to A2 under O2. Form A2 is less sensitive to O2 and may be converted into A1 under H2 atmosphere. Both active forms are present in aerobically isolated samples. Because the proteins are still active on 15% sodium dodecyl sulfate gels, they are not completely denatured, and the apparent molecular masses do not necessarily represent the true molecular masses of the enzymes. A1 has an Rf = 0.19, corresponding to an apparent molecular mass of 90 kDa, and A2 has an Rf = 0.35, corresponding to an apparent molecular mass of 49 kDa. A sedimentation equilibrium centrifugation study of the active enzyme shows that the holoenzyme has a molecular mass of 98 kDa. Form A2 may be separated into two subunits of molecular mass of 64 kDa and 34 kDa, respectively. Thus, form A2 represents the holoenzyme with a true molecular mass of 98 kDa. Amino acid compositions and N-terminal amino acid sequences of the A2 protein and these subunits are consistent with a heterodimeric holoenzyme. The relationship between the conformational changes detected in this study and a three-state scheme proposed on the basis of EPR spectroscopic studies of the metal-containing cofactors present in the enzyme is also discussed.     

Proteome analysis of the plant pathogen Xylella fastidiosa reveals major cellular and extracellular proteins and a peculiar codon bias distribution

The bacteria Xylella fastidiosa is the causative agent of a number of economically important crop diseases, including citrus variegated chlorosis. Although its complete genome is already sequenced, X. fastidiosa is very poorly characterized by biochemical approaches at the protein level. In an initial effort to characterize protein expression in X. fastidiosa we used one- and two-dimensional gel electrophoresis and mass spectrometry to identify the products of 142 genes present in a whole cell extract and in an extracellular fraction of the citrus isolated strain 9a5c. Of particular interest for the study of pathogenesis are adhesion and secreted proteins. Homologs to proteins from three different adhesion systems (type IV fimbriae, mrk pili and hsf surface fibrils) were found to be coexpressed, the last two being detected only as multimeric complexes in the high molecular weight region of one-dimensional electrophoresis gels. Using a procedure to extract secreted proteins as well as proteins weakly attached to the cell surface we identified 30 different proteins including toxins, adhesion related proteins, antioxidant enzymes, different types of proteases and 16 hypothetical proteins. These data suggest that the intercellular space of X. fastidiosa colonies is a multifunctional microenvironment containing proteins related to in vivo bacterial survival and pathogenesis. A codon usage analysis of the most expressed proteins from the whole cell extract revealed a low biased distribution, which we propose is related to the slow growing nature of X. fastidiosa. A database of the X. fastidiosa proteome was developed and can be accessed via the internet (URL: www.proteome.ibi.unicamp.br).     

A major single-stranded DNA binding protein from ovaries of the frog, Xenopus laevis, is lactate dehydrogenase

The most abundant single-stranded DNA binding protein (SSB) found in ovaries of the frog, Xenopus laevis, was purified to electrophoretic homogeneity. Under physiological conditions, the purified SSB lowered the Tm of poly[d(A-T)] and stimulated DNA synthesis by the homologous DNA polymerase DNA primase alpha complex on single-stranded DNA templates. These properties are characteristic of a bona fide single-stranded DNA binding protein. The Stokes radius of native SSB was calculated to be 45 A, corresponding to a molecular mass of about 140 kDa. On SDS polyacrylamide gels, the SSB migrated as a single band with a molecular mass of 36 kDa. We assumed, therefore, that the SSB was a tetramer of 36 kDa subunits. We subsequently discovered that the SSB was LDH, D-lactate dehydrogenase, EC 1.1.1.28. Purified SSB has high LDH specific activity. Following electrophoresis on SDS polyacrylamide gels, the 36 kDa subunits were renatured and exhibited LDH activity. The amino-acid composition of X. laevis SSB/LDH was similar to that of LDH from other species and to other reported single-stranded DNA binding proteins. Mammalian SSB/LDH also preferentially bound single-stranded DNA. Mammalian SSB/LDH bound to RNA as demonstrated by affinity chromatography on poly(A)-agarose and by its effect on translation of mRNA in vitro.     

Generating and navigating proteome maps using mass spectrometry

Proteomes, the ensembles of all proteins expressed by cells or tissues, are typically analysed by mass spectrometry. Recent technical and computational advances have greatly increased the fraction of a proteome that can be identified and quantified in a single study. Current mass spectrometry-based proteomic strategies have the potential to reproducibly, accurately, quantitatively and comprehensively measure any protein or whole proteomes from cells and tissues at different states. Achieving these goals will require complete proteome maps and analytical strategies that use these maps as prior information and will greatly enhance the impact of proteomics on biological and clinical research.     

Rice proteomics: a step toward functional analysis of the rice genome

The technique of proteome analysis with two-dimensional PAGE has the power to monitor global changes that occur in the protein expression of tissues and organisms and/or expression that occurs under stresses. In this study, the catalogues of the rice proteome were constructed, and a functional characterization of some of these proteins was examined. Proteins extracted from tissues of rice and proteins extracted from rice under various kinds of stress were separated by two-dimensional PAGE. An image analyzer was used to reveal a total of 10,589 protein spots on 10 kinds of two-dimensional PAGE gels stained by Coomassie Brilliant Blue. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane, and the N-terminal amino acid sequences of 272 of 905 proteins were determined. The internal amino acid sequences of 633 proteins were determined using a protein sequencer or mass spectrometry after enzyme digestion of the proteins. Finally, a data file of rice proteins that included information on amino acid sequences and sequence homologies was constructed. The major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that turned out to be calreticulin and a gibberellin-binding protein, which is ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have functions in the signal transduction pathway. The information thus obtained from the rice proteome will be helpful in predicting the function of the unknown proteins and will aid in their molecular cloning.     

Interrelation Between Protein Synthesis,Proteostasis and Life Span

The production of newly synthesized proteins is a key process of protein homeostasis that initiates the biosynthetic flux of proteins and thereby determines the composition, stability and functionality of the proteome. Protein synthesis is highly regulated on multiple levels to adapt the proteome to environmental and physiological challenges such as aging and proteotoxic conditions. Imbalances of protein folding conditions are sensed by the cell that then trigger a cascade of signaling pathways aiming to restore the protein folding equilibrium. One regulatory node to rebalance proteostasis upon stress is the control of protein synthesis itself. Translation is reduced as an immediate response to perturbations of the protein folding equilibrium that can be observed in the cytosol as well as in the organelles such as the endoplasmatic reticulum and mitochondria. As reduction of protein synthesis is linked to life span increase, the signaling pathways regu-lating protein synthesis might be putative targets for treatments of age-related diseases. Eukaryotic cells have evolved a complex system for protein synthesis regulation and this review will summarize cellular strategies to regulate mRNA translation upon stress and its impact on longevity.     

Myelin Proteomics: Molecular Anatomy of an Insulating Sheath

Fast-transmitting vertebrate axons are electrically insulated with multiple layers of nonconductive plasma membrane of glial cell origin, termed myelin. The myelin membrane is dominated by lipids, and its protein composition has historically been viewed to be of very low complexity. In this review, we discuss an updated reference compendium of 342 proteins associated with central nervous system myelin that represents a valuable resource for analyzing myelin biogenesis and white matter homeostasis. Cataloging the myelin proteome has been made possible by technical advances in the separation and mass spectrometric detection of proteins, also referred to as proteomics. This led to the identification of a large number of novel myelin-associated proteins, many of which represent low abundant components involved in catalytic activities, the cytoskeleton, vesicular trafficking, or cell adhesion. By mass spectrometry-based quantification, proteolipid protein and myelin basic protein constitute 17% and 8% of total myelin protein, respectively, suggesting that their abundance was previously overestimated. As the biochemical profile of myelin-associated proteins is highly reproducible, differential proteome analyses can be applied to material isolated from patients or animal models of myelin-related diseases such as multiple sclerosis and leukodystrophies.