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PubMed2004

Characterization and quantitation of membrane proteomes using multidimensional MS-based proteomic technologies.

A major goal of proteomics is to develop methods that enable the systematic characterization of every protein within the cell or particular subcellular proteome using a single analytical platform. Although the equivalent has already been achieved in genomics, reaching this goal in proteomics represents a much greater challenge due to the wide dynamic range of protein expression, numerous post-translational modifications and remarkable physicochemical heterogeneity of proteins. A major analytical challenge has involved developing more effective means for proteome-scale investigations of membrane proteins, whose solubility differs drastically from that of cytoplasmic proteins. Fortunately, rapid progress has increased the ability to characterize this critically important class of proteins on a scale analogous to that of aqueous soluble proteins.

Cell Adhesion↗
PubMed1998

The Australian Proteome Analysis Facility (APAF): assembling large scale proteomics through integration and automation.

The field of proteomics opens new possibilities for the mass screening of proteins from many different sources. While genomics is well understood to be a big science field, proteomics is just emerging as such. This paper describes the setting up of the first national proteomics facility. The facility has been funded by the Australian government and this funding has allowed the design of purpose built, integrated laboratories with state of the art equipment for large scale proteome research.

Academies and Institutes↗
PubMed2004

"Does understanding the brain need proteomics and does understanding proteomics need brains?"--Second HUPO HBPP Workshop hosted in Paris.

The second Human Brain Proteome Project (HBPP) Workshop of the Human Proteome Organisation (HUPO) took place at the Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI) from April 23-24, 2004. During two days, more than 70 attendees from Europe, Asia and the US came together to decide basic strategic approaches, standards and the beginning of a pilot phase prior to further studies of the human brain proteome. The international consortium presented the technological and scientific portfolio and scheduled the time table for the next year.

Animals↗
PubMed2005

Human Proteome Organisation Proteomics Standards Initiative. Pre-Congress Initiative.

The plenary session of the Proteomics Standards Initiative of the Human Proteome Organisation discussed the current status of the ongoing work in the fields of molecular interactions, mass spectrometry and the description of protein modifications. In addition, new areas are being opened up, in particular developing standards for the description and exchange of data from gel electrophoresis experiments. The General Proteomics Standards group is now working closely with the Functional Genomics Experiment efforts to define a general standard in which to encode data that will enable a systems biology approach to data analysis.

Databases, Genetic↗
PubMed2007

Evaluation of two proteomics technologies used to screen the membrane proteomes of wild-type Corynebacterium glutamicum and an L-lysine-producing strain.

The membrane proteomes of a wild-type Corynebacterium glutamicum and an L-lysine-producing strain were quantitatively analyzed by two complementary proteomics techniques -- anion exchange chromatography AIEC/SDS-PAGE and 16BAC-PAGE/SDS-PAGE -- and the results were compared. Although both techniques allow for the fast screening of differences in protein abundance, AIEC/SDS-PAGE was superior to 16BAC-PAGE/SDS-PAGE with respect to protein separation, it was more suitable for relative protein quantification, and allowed more differentially regulated proteins to be detected (the succinate dehydrogenase complex, an ABC-type cobalamin/Fe(3+) siderophore transport system, the maltose binding protein, and a subunit of the cytochrome bc-aa(3) supercomplex were upregulated, while a periplasmic component of an ABC-type transporter and an iron-regulated ABC-type transporter were downregulated in the producer). The results indicate the important role of tricarboxylic acid cycle enzymes as well as the adaptation of transport processes in L-lysine-producing cells. Since the only genetic differences between the wild type and the L-lysine producer occur between four central metabolic enzymes in the cytoplasm, our study illustrates the complex effects of metabolic engineering on cell physiology and the power of the new AIEC/SDS-PAGE proteomics approach to detect these effects.

ATP-Binding Cassette Transporters↗
PubMedDate not supplied

Mammalian embryonic cerebrospinal fluid proteome has greater apolipoprotein and enzyme pattern complexity than the avian proteome.

During early stages of embryo development, the brain cavity is filled with Embryonic Cerebro-Spinal Fluid, which has an essential role in the survival, proliferation and neurogenesis of the neuroectodermal stem cells. We identified and analyzed the proteome of Embryonic Cerebro-Spinal Fluid from rat embryos (Rattus norvegicus), which includes proteins involved in the regulation of Central Nervous System development. The comparison between mammalian and avian Embryonic Cerebro-Spinal Fluid proteomes reveals great similarity, but also greater complexity in some protein groups. The pattern of apolipoproteins and enzymes in CSF is more complex in the mammals than in birds. This difference may underlie the greater neural complexity and synaptic plasticity found in mammals. Fourteen Embryonic Cerebro-Spinal Fluid gene products were previously identified in adult human Cerebro-Spinal Fluid proteome, and interestingly they are altered in patients with neurodegenerative diseases and/or neurological disorders. Understanding these molecules and the mechanisms they control during embryonic neurogenesis may contribute to our understanding of Central Nervous System development and evolution, and these human diseases.

Animals↗
PubMed2006

A new functional, chemical proteomics technology to identify purine nucleotide binding sites in complex proteomes.

Adenine nucleotides are small, abundant molecules that bind numerous proteins involved in pivotal cellular processes. These nucleotides are co-factors or substrates for enzymes, regulators of protein function, or structural binding motifs. The identification of nucleotide-binding sites on a proteome-wide scale is tempting in view of the high number of nucleotide-binding proteins, their large in vivo concentration differences, and the various functions they exert. Here, we report on a functional, chemical, gel-free proteomics technology that allows the identification of protein adenine nucleotide-binding site(s) in cell lysates. Our technology uses a synthetic ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), as an affinity/activity-based probe for nucleotide-binding sites. When applied on a cellular level, 185 different FSBA-labeled sites in a human Jurkat cell lysate were identified. Functional and structural aspects of the use of FSBA on a proteome-wide scale are discussed.

Adenine↗
PubMed2006

Systematic characterization of nuclear proteome during apoptosis: a quantitative proteomic study by differential extraction and stable isotope labeling.

Identification and characterization of the nuclear proteome is important for detailed understanding of multiple signaling events in eukaryotic cells. Toward this goal, we extensively characterized the nuclear proteome of human T leukemia cells by sequential extraction of nuclear proteins with different physicochemical properties using three buffer conditions. This large scale proteomic study also tested the feasibility and technical challenges associated with stable isotope labeling by amino acids in cell culture (SILAC) to uncover quantitative changes during apoptosis. Analyzing proteins from three nuclear fractions extracted from naive and apoptotic cells generated 780,530 MS/MS spectra that were used for database searching using the SEQUEST algorithm. This analysis resulted in the identification and quantification of 1,174 putative nuclear proteins. A number of known nuclear proteins involved in apoptosis as well as novel proteins not known to be part of the nuclear apoptotic machinery were identified and quantified. Consistent with SILAC-based quantifications, immunofluorescence staining of nucleus, mitochondria, and some associated proteins from both organelles revealed a dynamic recruitment of mitochondria into nuclear invaginations during apoptosis.

Amino Acid Sequence↗
PubMed2006

Extent of modifications in human proteome samples and their effect on dynamic range of analysis in shotgun proteomics.

The complexity of the human proteome, already enormous at the organism level, increases further in the course of the proteome analysis due to in vitro sample evolution. Most of in vitro alterations can also occur in vivo as post-translational modifications. These two types of modifications can only be distinguished a posteriori but not in the process of analysis, thus rendering necessary the analysis of every molecule in the sample. With the new software tool ModifiComb applied to MS/MS data, the extent of modifications was measured in tryptic mixtures representing the full proteome of human cells. The estimated level of 8-12 modified peptides per each unmodified tryptic peptide present at >or=1% level is approaching one modification per amino acid on average. This is a higher modification rate than was previously thought, posing an additional challenge to analytical techniques. The solution to the problem is seen in improving sample preparation routines, introducing dynamic range-adjusted thresholds for database searches, using more specific MS/MS analysis using high mass accuracy and complementary fragmentation techniques, and revealing peptide families with identification of additional proteins only by unfamiliar peptides. Extensive protein separation prior to analysis reduces the requirements on speed and dynamic range of a tandem mass spectrometer and can be a viable alternative to the shotgun approach.

Amino Acid Sequence↗
PubMed2005

The dynamics of the proteome: strategies for measuring protein turnover on a proteome-wide scale.

Quantitative proteomics captures the steady-state amount of a protein in a cell but does not explain how a change in protein amount is manifest -- whether through a change in synthesis or a change in degradation. If we are to understand the changes in the proteome, we will need to define such processes. In this brief review, strategies for the determination of intracellular protein dynamics on a proteome-wide scale are discussed.

Animals↗
PubMed2003

Organelle proteomics: implications for subcellular fractionation in proteomics.

Functional proteome analysis is not restricted to the sequence information but includes the broad spectrum of structural modifications and quantitative changes of proteins to which they are subjected in different tissues and cell organelles and during the development of an organism. Cell biology has provided the means required for the analysis of the composition and properties of purified cellular elements. Subcellular fractionation is an approach universal across all cell types and tissues, including cardiac and vascular system. Subcellular fractionation and proteomics form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. Subcellular fractionation is a flexible and adjustable approach resulting in reduced sample complexity and is most efficiently combined with high-resolution 2D gel/mass spectrometry analysis as well as with gel-independent techniques. In this study we introduce state of the art subcellular fractionation techniques and discuss their suitability, advantages, and limitations for proteomics research.

Animals↗
PubMed2006

Breast cancer proteomics using two-dimensional electrophoresis: studying the breast cancer proteome.

Proteomics has emerged as a powerful approach for studying disease-associated changes in protein levels. The most commonly used method in proteomics studies remains two-dimensional gel electrophoresis, but the methodology, standardization, and interpretation of the results obtained require considerable expertise. In this chapter, we describe the approaches we have taken to studying the breast cancer proteome, using cells grown in vitro and cancer specimens as the starting materials.

Animals↗
PubMed2006

Chemistry meets proteomics: the use of chemical tagging reactions for MS-based proteomics.

As proteomics matures from a purely descriptive to a function-oriented discipline of the life sciences, there is strong demand for novel methodologies that increase the depth of information that can be obtained from proteomic studies. MS has long played a central role for protein identification and characterization, often in combination with dedicated chemical modification reactions. Today, chemistry is helping to advance the field of proteomics in numerous ways. In this review, we focus on those methodologies that have a significant impact for the large-scale study of proteins and peptides. This includes approaches that allow the introduction of affinity tags for the enrichment of subclasses of peptides or proteins and strategies for in vitro stable isotope labeling for quantification purposes, among others. Particular attention is given to the study of PTMs where recent advancements have been promising, but many interesting targets are not yet being addressed.

Animals↗
PubMed2006

Integrative proteomics: structure, function, and interaction report on the 3rd joint meeting of the British Society for Proteome Research and the European Bioinformatics Institute, July 2006.

This report summarizes the highlights of the recent British Society for Proteome Research (BSPR) meeting jointly organized with the European Bioinformatics Institute (EBI) which was held at the Wellcome Trust Genome Campus, Hinxton, Cambridge, UK in July 2006. This was the third annual scientific meeting organized by the BSPR and EBI and the theme of this years meeting was Integrative Proteomics: Structure, function and interaction. A wealth of local and overseas speakers were invited to discuss both their own work and specific challenges present in modern day proteomic based experiments.

Computational Biology↗
PubMed2006

Activity-based proteomics: enzymatic activity profiling in complex proteomes.

In the postgenomic era new technologies are emerging for global analysis of protein function. The introduction of active site-directed chemical probes for enzymatic activity profiling in complex mixtures, known as activity-based proteomics has greatly accelerated functional annotation of proteins. Here we review probe design for different enzyme classes including serine hydrolases, cysteine proteases, tyrosine phosphatases, glycosidases, and others. These probes are usually detected by their fluorescent, radioactive or affinity tags and their protein targets are analyzed using established proteomics techniques. Recent developments, such as the design of probes for in vivo analysis of proteomes, as well as microarray technologies for higher throughput screenings of protein specificity and the application of activity-based probes for drug screening are highlighted. We focus on biological applications of activity-based probes for target and inhibitor discovery and discuss challenges for future development of this field.

Animals↗
PubMed2007

Multiplexed proteomic reactor for the processing of proteomic samples.

We report the development of a 96-well plate proteomic reactor for gel-free processing of minute amounts of complex proteomic samples. The device performs multiplexed trapping, enrichment, and biochemical processing of proteins, resulting in concentrated peptide solutions ready for mass spectrometric analysis. Individual wells on the reactor can process up to 2 microg of protein. We also report the coupling of the plate proteomic reactor with protein fractionation using size-exclusion chromatography for large-scale identification of proteins. To illustrate the potential of this approach, we separated 400 microg of MCF7 cell lysate using size-exclusion chromatography and processed 35 protein fractions on the reactor plate. Using stringent criteria when searching the data, a total of 875 unique proteins were identified. More relaxed searching conditions associated with a 1% false positive rate led to the identification of 2683 unique proteins, meaning that one protein was identified per 3-10 ng of total protein lysate loaded on the reactor plate.

Biomarkers, Tumor↗
PubMed2006

Computational Proteomics Analysis System (CPAS): an extensible, open-source analytic system for evaluating and publishing proteomic data and high throughput biological experiments.

The open-source Computational Proteomics Analysis System (CPAS) contains an entire data analysis and management pipeline for Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) proteomics, including experiment annotation, protein database searching and sequence management, and mining LC-MS/MS peptide and protein identifications. CPAS architecture and features, such as a general experiment annotation component, installation software, and data security management, make it useful for collaborative projects across geographical locations and for proteomics laboratories without substantial computational support.

Computational Biology↗
PubMed2006

High light response of the thylakoid proteome in arabidopsis wild type and the ascorbate-deficient mutant vtc2-2. A comparative proteomics study.

The thylakoid proteome of chloroplasts contains multiple proteins involved in antioxidative defense, protein folding, and repair. To understand this functional protein network, we analyzed the quantitative response of the thylakoid-associated proteome of Arabidopsis (Arabidopsis thaliana) wild type and the ascorbate-deficient mutant vtc2-2 after transition to high light (HL; 1,000 micromol photons m(-2) s(-1)). The soluble thylakoid proteomes of wild type and vtc2-2 were compared after 0, 1, 3, and 5 d of HL using two-dimensional gels with three independent experiments, followed by a multivariant statistical analysis and tandem mass spectrometry. After 5 d of HL, both wild-type and vtc2-2 plants accumulated anthocyanins, increased their total ascorbate content, and lost 10% of photosystem II efficiency, but showed no bleaching. Anthocyanin and total ascorbate concentrations in vtc2-2 were respectively 34% and 20% of wild type, potentially leading to enhanced oxidative stress in vtc2-2. Forty-five protein spots significantly changed as a consequence of genotype, light treatment, or both. Independent confirmation was obtained from western blots. The most significant response was the up-regulation of thylakoid YCF37 likely involved in photosystem I assembly, and specific fibrillins, a flavin reductase-like protein, and an aldolase, each located in thylakoid-associated plastoglobules. Fe-superoxide dismutase was down-regulated in vtc2-2, while Cu,Zn-superoxide dismutase was up-regulated. vtc2-2 also showed a systematic up-regulation of a steroid dehydrogenase-like protein. A number of other stress-related proteins, several thylakoid proteases, and lumenal isomerases did not change, while PsbS increased in wild type upon light stress. These findings are discussed in terms of plastid metabolism and oxidative stress defense, and emphasize that understanding of the chloroplast stress-response network must include the enzymatic role of plastoglobules.

Arabidopsis↗