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PubMed2001

From proteomics to genomics.

Presently, science is moving from genomics to proteomics in order to get insight into the functional network of gene expression. Actually however, proteomics is much older than genomics and dates back to the introduction of the two-dimensional gel electrophoresis technique (2-DE) independently by Klose and O'Farrell. Based on this approach almost all cellular proteins can be separated. New developments in mass spectrometry allowed identification of single spots in the 2-DE protein pattern, including the underlying genes. Joachim Klose has focused his pioneering 2-DE studies on mouse models with special emphasis on quantitative protein variants. According to him, proteins are living molecules exhibiting a characteristic protein phenotype.

Animals↗
PubMed2000

Proteomics approach in classifying the biochemical basis of the anticancer activity of the new olomoucine-derived synthetic cyclin-dependent kinase inhibitor, bohemine.

The aim of this study was to use two-dimensional electrophoresis (2-DE) coupled with multivariate principal component analysis (PCA) to characterize the quantitative changes in the protein composition of the CEM T-lymphoblastic leukemia cell line after treatment with bohemine (BOH), a synthetic olomoucin-derived cyclin-dependent kinase inhibitor (CDKI). Cell classification, reflecting protein patterns, clearly distinguished two main groups: one group consists of 9, 12 and 24 h treated BOH cells while the second is represented by the 0 and 24 h control untreated cells and the 6 h BOH-exposed CEM lymphoblasts. Discriminant protein spots differentially expressed in the BOH-treated CEM cells were selected for identification by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or electrospray ionization-tandem MS (ESI-MS/MS). Five of the selected protein spots were unequivocally identified as alpha-enolase, triosephosphate isomerase, eukaryotic initiation factor 5A, and alpha- and beta-subunits of Rho GDP-dissociation inhibitor 1. These proteins, all significantly downregulated in CEM T-lymphoblast leukemia in the course of BOH treatment, are known to play an important role in cellular functions such as glycolysis, protein biosynthesis, and cytoskeleton rearrangement. These results indicate that the cellular effects of olomoucine-derived CDKIs are not dependent on their ability to inhibit CDKs and could be mediated by several factors such as a decrease in protein synthesis and/or glycolysis which in turn diminishes the ability of cancer cells to function.

Antineoplastic Agents↗
PubMed2002

Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system.

The technique of fluorescent two-dimensional (2D) difference gel electrophoresis for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 overexpression. Labeling of paired cell lysate samples with N-hydroxy succinimidyl ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive, and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrix-assisted laser-desorption ionization mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3- and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (Amersham Biosciences, Inc.) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines.

Breast Neoplasms↗
PubMedDate not supplied

Perspective: a program to improve protein biomarker discovery for cancer.

Biomarkers for cancer risk, early detection, prognosis, and therapeutic response promise to revolutionize cancer management. Protein biomarkers offer tremendous potential in this regard due to their great diversity and intimate involvement in physiology. An effective program to discover protein biomarkers using existing technology will require team science, an integrated informatics platform, identification and quantitation of candidate biomarkers in disease tissue, mouse models of disease, standardized reagents for analyzing candidate biomarkers in bodily fluids, and implementation of automation. Technology improvements for better fractionation of the proteome, selection of specific biomarkers from complex mixtures, and multiplexed assay of biomarkers would greatly enhance progress.

Animals↗
PubMed2006

The lipid-droplet proteome reveals that droplets are a protein-storage depot.

BACKGROUND: Lipid droplets are ubiquitous organelles that are among the basic building blocks of eukaryotic cells. Despite central roles for cholesterol homeostasis and lipid metabolism, their function and protein composition are poorly understood. RESULTS: We purified lipid droplets from Drosophila embryos and analyzed the associated proteins by capillary LC-MS-MS. Important functional groups include enzymes involved in lipid metabolism, signaling molecules, and proteins related to membrane trafficking. Unexpectedly, histones H2A, H2Av, and H2B were present. Using biochemistry, genetics, real-time imaging, and cell biology, we confirm that roughly 50% of certain embryonic histones are physically attached to lipid droplets, a localization conserved in other fly species. Histone association with droplets starts during oogenesis and is prominent in early embryos, but it is undetectable in later stages or in cultured cells. Histones on droplets are not irreversibly trapped; quantitation of droplet histone levels and transplantation experiments suggest that histones are transferred from droplets to nuclei as development proceeds. When this maternal store of histones is unavailable because lipid droplets are mislocalized, zygotic histone production starts prematurely. CONCLUSIONS: Because we uncover a striking proteomic similarity of Drosophila droplets to mammalian lipid droplets, Drosophila likely provides a good model for understanding droplet function in general. Our analysis also reveals a new function for these organelles; the massive nature of histone association with droplets and its developmental time-course suggest that droplets sequester maternally provided proteins until they are needed. We propose that lipid droplets can serve as transient storage depots for proteins that lack appropriate binding partners in the cell. Such sequestration may provide a general cellular strategy for handling excess proteins.

Animals↗
PubMed2006

Proteomic signature of human embryonic stem cells.

Human embryonic stem cells (hESC) represent a population of undifferentiated pluripotent cells with both self-renewal and multilineage differentiation characteristics. Proteomics provides a powerful approach for studying the characteristics of hESC and discovering molecular markers. We have analyzed proteome profiles of three hESC lines using 2-DE and MALDI TOF-TOF. Out of 844 spots analyzed with MALDI TOF-TOF, 685 proteins were identified of which 60 proteins were classified as the most abundant proteins on 2-D gels. A large number of proteins particularly high abundant ones were identified as chaperones, heat shock proteins, ubiquitin/proteasome, and oxidative stress responsive proteins underscoring the ability of these cells to resist oxidative stress and increase the life span. Several proteins involved in cell proliferation and differentiation were also among the highly expressed proteins. Although overall expression pattern of three hESC were similar, 54 spots changed quantitatively and 14 spots changed qualitatively among the hESC cell lines. Most of these proteins were identified as proteins involved in cell growth, metabolism and signal transduction, which may affect the self-renewal and pluripotency. To our knowledge, this study represents the first proteomic dataset for hESC and provides a better insight into the biology of hESC. Proteome maps of hESC are accessible at http://www.RoyanProteomics.ir.

Cell Line↗
PubMed2006

NOPdb: Nucleolar Proteome Database.

The Nucleolar Proteome Database (NOPdb) archives data on >700 proteins that were identified by multiple mass spectrometry (MS) analyses from highly purified preparations of human nucleoli, the most prominent nuclear organelle. Each protein entry is annotated with information about its corresponding gene, its domain structures and relevant protein homologues across species, as well as documenting its MS identification history including all the peptides sequenced by tandem MS/MS. Moreover, data showing the quantitative changes in the relative levels of approximately 500 nucleolar proteins are compared at different timepoints upon transcriptional inhibition. Correlating changes in protein abundance at multiple timepoints, highlighted by visualization means in the NOPdb, provides clues regarding the potential interactions and relationships between nucleolar proteins and thereby suggests putative functions for factors within the 30% of the proteome which comprises novel/uncharacterized proteins. The NOPdb (http://www.lamondlab.com/NOPdb) is searchable by either gene names, nucleotide or protein sequences, Gene Ontology terms or motifs, or by limiting the range for isoelectric points and/or molecular weights and links to other databases (e.g. LocusLink, OMIM and PubMed).

Cell Nucleolus↗
PubMed2004

Proteomic analysis of protein changes developing in rat hippocampus after chronic antidepressant treatment: Implications for depressive disorders and future therapies.

It is recognized that monoamine reuptake inhibitors (MARIs) exert beneficial effects in the treatment of major depression and general anxiety disorder. The aim of this study was to identify proteins regulated by this class of antidepressant using a proteome differential profiling approach. Either venlafaxine or fluoxetine was administered systemically to adult rats for 2 weeks, and protein patterns from rat hippocampal cytosolic extracts were compared by two-dimensional gel electrophoresis. Silver-stained protein spots displaying differential expression were identified by mass spectrometry. Thirty-three protein spots were modulated by both drug treatments compared to controls. The classification of several proteins that were sorted by function suggested convergent pathway activities for both MARIs at the post-receptor level. These included proteins associated with neurogenesis (insulin like growth factor 1 (IGF-1), glia maturation factor [GMF]-beta), outgrowth/maintenance of neuronal processes (hippocampal cholinergic neurostimulating peptide [HCNP], PCTAIRE-3), and with neural regeneration/axonal guidance collapsin response mediator protein (CRMP-2) systems. Other modulated proteins indicated an increase in neuronal vesicular cell trafficking and synaptic plasticity (Ras-related protein 4a (Rab4a), Ras-related protein 1b (Rab1b), heat shock protein 10 [HSP10]), as well as neurosteroidogenic (hydroxysteroid sulfotransferase A) and possible anti-apoptotic (dimethylargininase-1 L-N,N-dimethylarginine dimethylaminohydrolase-1 [DDAH-1], pyruvate dehydrogenase-E1 [PDH-E1], antioxidant protein-2 [AOP-2]) pathway-mediated regulatory events. Parallel studies to investigate further the effects of venlafaxine and fluoxetine on adult hippocampal neurogenesis in vivo by quantitative bromodeoxyuridine immunolabeling revealed a significant drug-induced increase in the proliferation rate and long-term survivability of progenitor stem cells located in the subgranular zone. These data suggest that MARIs share wide-ranging proteome changes within the hippocampal formation, beyond 5-HT/NE neurotransmission. This may reflect long-term functional adaptations required for antidepressant activity.

Animals↗
PubMed2004

N-t-butyliodoacetamide and iodoacetanilide: two new cysteine alkylating reagents for relative quantitation of proteins.

The synthesis and application of two new alkylating reagents, N-tert-butyl-2-iodoacetamide (N-t-butyliodoacetamide) and 2-iodo-N-phenylacetamide (iodoacetanilide), are described. N-t-Butyliodoacetamide and iodoacetanilide were synthesised to purity in their d(0)-light and in their respective d(9)- and d(5)-heavy forms. The newly synthesised reagents are covalently bound to peptides containing cysteines via an alkylation reaction. The mass differences of 5 and 9 Da avoid possible problems of overlapping isotope distribution. For each alkylated cysteine a peptide mass increases, respectively, by a multiple of 113 and 133 Da for the d(0)-light form of N-t-butyliodoacetamide and iodoacetanilide. These reagents can therefore replace common alkylating reagents in existing proteomics-based applications. Alkylated peptides increase in mass in the same mass range as amino acids and remain suitable for tandem mass spectrometry (MS/MS) data acquisition and analysis. The compounds are simple to use and derivatisation is based on widely applied alkylating procedures. Preliminary results show that these reagents can be applied for both protein quantitation and identification by peptide mass finger printing and/or MS/MS techniques. Using these chemicals and the suggested workflow enables the quantitative analysis of the whole protein sample and realises access to peptides that may contain potential post-translational modifications. Other approaches that incorporate a matrix-assisted laser desorption/ionisation (MALDI) interface prior to MS can take advantage of these chemicals, such as the molecular scanner.

Acetanilides↗
PubMed2001

Possibilities to improve automation, speed and precision of proteome analysis: a comparison of two-dimensional electrophoresis and alternatives.

Proteome analysis requires fast methods with high separation efficiencies in order to screen the various cell and tissue types for their proteome expression and monitor the effect of environmental conditions and time on this expression. The established two-dimensional gel electrophoresis (2-DE) is by far too slow for a consequential screening. Moreover, it is not precise enough to observe changes in protein concentrations. There are various approaches that promise faster, automated proteome analysis. This article concentrates on capillary (CT isoelectric focusing coupled to mass spectrometry (CIEF-MSn) and preparative IEF followed by size-exclusion chromatography, hyphenated with MS (PIEF-SEC-MS). These two approaches provide a similar separation pattern as the established 2-DE technique and therefore allow for the continued use of data based on this traditional approach. Their performances have been discussed and compared to 2-DE, evaluating 169 recent articles. Data on analysis time, automation, the detection limit, quantitation, peak capacity, mass and pI accuracy, as well as on the required sample amount are compared in a table.

Automation↗
PubMed2006

Searching for aging-related proteins in human dermal microvascular endothelial cells treated with anti-aging agents.

Endothelial cells constitute an interface between blood and tissue and act as a medium for active interaction between plasma and the intracellular environment for homeostasis. Aging of endothelial cells plays a significant role in the pathophysiology of age-related vascular diseases; however, precise mechanisms for senescence have not been elucidated. Proteomics allows identification of protein structures, functions, and characteristics, and can be applied to the study of aging processes. Using cultured human dermal microvascular endothelial cells and two-dimensional proteomic mapping, we studied the effects of kinetin, epigallocatechin-3-gallate, all-trans-retinoic acid, and selenium on their senescence and searched for the aging-related proteins. The treatments resulted in 68 qualitative changes and 172 quantitative changes, and we were able to identify 46 spots among them. All of the agents indicated above induced changes in the expression of moesin, rho guanosine-5'-diphosphate-dissociation inhibitor, and actin, confirmed by immunoblotting and confocal laser microscopy. As these proteins were associated with cell cycle and cytoskeleton, immunoblotting of the proteins related to cell cycle was performed. Although practical significance remains to be confirmed by in vivo research, this fundamental discovery may provide a basis for understanding the mechanism of aging and age-related diseases.

Aging↗
PubMed2006

Deviation from the mobile proton model in amino-modified peptides: implications for multiple reaction monitoring analysis of peptides.

The study of peptide fragmentation is important to the understanding of chemical processes occurring in the gas phase and the more practical concern of peptide identification for proteomic analysis. Using the mobile proton model as a framework, we explore the effect of amino-group modifications on peptide fragmentation. Three aldehydes are used to transform the peptides' primary amino groups into either a dimethylamino or a heterocyclic structure (five- or six-membered). The observed fragmentation patterns deviate strongly from those observed for the analogous underivatised peptides. In particular, the a1 ion is the base peak in most tandem mass spectra of the derivatised peptides. The a1 ion intensity depends strongly on the N-terminal amino acid, with tyrosine and phenylalanine having the strongest enhancement. Despite the change in fragmentation patterns of the derivatised peptides, they still provide high-quality tandem mass spectra that, in many cases, are more amenable to database searching than the spectra of underivatised peptides. In addition, the reliable presence of the a1 ion facilitates rapid quantitative measurements using the multiple reaction monitoring approach.

Aldehydes↗
PubMed2006

Numerous and rapid nonstochastic modifications of gene products in newly synthesized Brassica napus allotetraploids.

Polyploidization is a widespread process that results in the merger of two or more genomes in a common nucleus. To investigate modifications of gene expression occurring during allopolyploid formation, the Brassica napus allotetraploid model was chosen. Large-scale analyses of the proteome were conducted on two organs, the stem and root, so that >1600 polypeptides were screened. Comparative proteomics of synthetic B. napus and its homozygous diploid progenitors B. rapa and B. oleracea showed that very few proteins disappeared or appeared in the amphiploids (<1%), but a strikingly high number (25-38%) of polypeptides displayed quantitative nonadditive pattern. Nonstochastic gene expression repatterning was found since 99% of the detected variations were reproducible in four independently created amphiploids. More than 60% of proteins displayed a nonadditive pattern closer to the paternal parent B. rapa. Interspecific hybridization triggered the majority of the deviations (89%), whereas very few variations (approximately 3%) were associated with genome doubling and more significant alterations arose from selfing (approximately 9%). Some nonadditive proteins behaved similarly in both organs, while others exhibited contrasted behavior, showing rapid organ-specific regulation. B. napus formation was therefore correlated with immediate and directed nonadditive changes in gene expression, suggesting that the early steps of allopolyploidization repatterning are controlled by nonstochastic mechanisms.

Biological Evolution↗
PubMed2006

Alterations in the mitochondrial proteome of adriamycin resistant MCF-7 breast cancer cells.

A 2D-gel based comparative proteomic analysis has been conducted of changes in mitochondrial protein abundances in MCF-7 human breast cancer cells selected for resistance to Adriamycin accompanied by verapamil. We identified and compared 156 unique proteins from 184 spots. Eleven mitochondrial proteins were found with abundances altered more than 2-fold. Transcription was evaluated for two of these, using quantitative RT-PCR. Implications of the changes are considered with respect to drug resistance.

Breast Neoplasms↗
PubMed2005

Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis.

BACKGROUND: The analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids. RESULTS: After initial analyses by one-dimensional SDS-PAGE, quantitative two-dimensional analyses of human erythrocyte membranes, mouse liver membranes, and mouse brain membranes, extracted with buffers that included the zwitterionic detergent MEGA 10 (decanoyl-N-methylglucamide) and the zwitterionic lipid LPC (1-lauroyl lysophosphatidylcholine), showed selective improvement over extraction with the common 2-DE detergent CHAPS (3 [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Mixtures of the three detergents showed additive improvements in spot number, density, and resolution. Substantial improvements in the analysis of a brain membrane proteome were observed. CONCLUSION: This study demonstrates that an optimized detergent mix, coupled with rigorous sample handling and electrophoretic protocols, enables simple and effective analysis of membrane proteomes using two-dimensional electrophoresis.

Journal Article↗
PubMed2004

Proteomics as a tool to improve investigation of substantial equivalence in genetically modified organisms: the case of a virus-resistant tomato.

At present, the so-called "substantial equivalence" is the only widely accepted criterion for deciding whether or not a transgenic food is, from an alimentary point of view, to be considered totally correspondent to the "traditional" one from which it derives. Although never exactly defined, it deals with a comparison between the chemical composition of the two foods. A more in-depth analysis can be performed by one of the most suitable methods that allows for the simultaneous screening of many components without prior identification, the analysis of the proteome. As a model for testing this kind of approach, we compared protein expression of two types of tomato plants, having the same genetic background, except for a virus resistance trait introduced by genetic engineering. When proteins extracted from seedlings of the two types were analyzed by two-dimensional electrophoresis, no significant differences, either qualitative or quantitative, were detected, indicating that in this case the expression of major proteins was unmodified by the genetic manipulation. Fifteen proteins were identified by peptide mass fingerprinting.

Solanum lycopersicum↗
PubMed2006

Molecular imaging promotes progress in orthopedic research.

Modern orthopedic research is directed towards the understanding of molecular mechanisms that determine development, maintenance and health of musculoskeletal tissues. In recent years, many genetic and proteomic discoveries have been made which necessitate investigation under physiological conditions in intact, living tissues. Molecular imaging can meet this demand and is, in fact, the only strategy currently available for noninvasive, quantitative, real-time biology studies in living subjects. In this review, techniques of molecular imaging are summarized, and applications to bone and joint biology are presented. The imaging modality most frequently used in the past was optical imaging, particularly bioluminescence and near-infrared fluorescence imaging. Alternate technologies including nuclear and magnetic resonance imaging were also employed. Orthopedic researchers have applied molecular imaging to murine models including transgenic mice to monitor gene expression, protein degradation, cell migration and cell death. Within the bone compartment, osteoblasts and their stem cells have been investigated, and the organic and mineral bone phases have been assessed. These studies addressed malignancy and injury as well as repair, including fracture healing and cell/gene therapy for skeletal defects. In the joints, molecular imaging has focused on the inflammatory and tissue destructive processes that cause arthritis. As described in this review, the feasibility of applying molecular imaging to numerous areas of orthopedic research has been demonstrated and will likely result in an increase in research dedicated to this powerful strategy. Molecular imaging holds great promise in the future for preclinical orthopedic research as well as next-generation clinical musculoskeletal diagnostics.

Animals↗
PubMed2005

The application of inductively coupled plasma mass spectrometry in pharmaceutical and biomedical analysis.

With the development of life science, pharmaceutical and biomedical analysis becomes more and more important in medical science. Further studies will be hopefully established if it is possible to use inorganic elemental standards or small organic compounds in the quantitative determination of all kinds of drugs, nucleotides and sulfur or phosphorus containing peptides and proteins at appropriate concentration with an acceptable accuracy. Since 1980, inductively coupled plasma mass spectrometry (ICP-MS) has emerged as a new and powerful analytical technique which is suitable for element and isotope analysis. It offers extremely wide detection range of element and co-analysis of most elements in the periodic table. Also, it can be applied to perform qualitative, semiquantitative, and quantitative analysis and isotopic ratios through mass-to-electric charge ratio. With the help of ICP-MS, the struggle of searching for an excellent quantification technique in, e.g. drugs and proteomics has come appreciably close to an end. This review mainly focuses on the introduction of application of ICP-MS in pharmaceutical and biomedical analysis. Some problems in application and the handling strategies are simply presented at the end.

Biomedical Technology↗