Quantitative proteomics using uniform 15N-labeling, mascot and the trans-proteomic pipeline

Stable isotope labeling combined with MS is a powerful method for measuring relative protein abundances, for instance, by differential metabolic labeling of some or all amino acids with 14N and 15N in cell culture or hydroponic media. These and most other types of quantitative proteomics experiments using high-throughput technologies, such as LC-MS/MS, generate large amounts of raw MS data. This data needs to be processed efficiently and automatically, from the mass spectrometer to statistically evaluated protein identifications and abundance ratios. This paper describes in detail an approach to the automated analysis of uniformly 14N/15N-labeled proteins using MASCOT peptide identification in conjunction with the trans-proteomic pipeline (TPP) and a few scripts to integrate the analysis workflow. Two large proteomic datasets from uniformly labeled Arabidopsis thaliana were used to illustrate the analysis pipeline. The pipeline can be fully automated and uses only common or freely available software.

Eukaryotic expression: developments for structural proteomics

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe ( SPINE) consortium have developed and implemented high- throughput ( HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast ( Pichia pastoris and Saccharomyces cerevisiae), baculovirusinfected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for coexpression, selenomethionine labelling ( in all three eukaryotic systems) and control of glycosylation ( for secreted proteins in mammalian cells) are assessed.

High-throughput proteomics using matrix-assisted laser desorption/ ionization mass spectrometry

It has become evident that the mystery of life will not be deciphered just by decoding its blueprint, the genetic code. In the life and biomedical sciences, research efforts are now shifting from pure gene analysis to the analysis of all biomolecules involved in the machinery of life. One area of these postgenomic research fields is proteomics. Although proteomics, which basically encompasses the analysis of proteins, is not a new concept, it is far from being a research field that can rely on routine and large-scale analyses. At the time the term proteomics was coined, a gold-rush mentality was created, promising vast and quick riches (i.e., solutions to the immensely complex questions of life and disease). Predictably, the reality has been quite different. The complexity of proteomes and the wide variations in the abundances and chemical properties of their constituents has rendered the use of systematic analytical approaches only partially successful, and biologically meaningful results have been slow to arrive. However, to learn more about how cells and, hence, life works, it is essential to understand the proteins and their complex interactions in their native environment. This is why proteomics will be an important part of the biomedical sciences for the foreseeable future. Therefore, any advances in providing the tools that make protein analysis a more routine and large-scale business, ideally using automated and rapid analytical procedures, are highly sought after. This review will provide some basics, thoughts and ideas on the exploitation of matrix-assisted laser desorption/ ionization in biological mass spectrometry - one of the most commonly used analytical tools in proteomics - for high-throughput analyses.

In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria graminis f. sp hordei

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. Molecular & Cellular Proteomics 8: 2368-2381, 2009.

Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study

Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as N-15. By utilising hydroponic media that contain N-15 inorganic salts as the sole nitrogen source, near to 100% N-15-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled N-14- and N-15-hydroponically grown plants were fractionated by SDS-PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Proteins were identified and the spectra of N-14/N-15 peptide pairs were extracted using their m/z chromatographic retention time, isotopic distributions, and the m/z difference between the N-14 and N-15 peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct N-14 and N-15 peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2). (C) 2008 Elsevier Ltd. All rights reserved.

Combinatorial peptide ligand libraries and plant proteomics: a winning strategy at a price

The mechanism of action and properties of a solid-phase ligand library made of hexapeptides (combinatorial peptide ligand libraries or CPLL, for capturing the "hidden proteome", i.e. the low- and very low-abundance proteins Constituting the vast majority of species in any proteome. as applied to plant tissues, are reviewed here. Plant tissues are notoriously recalcitrant to protein extraction and to proteome analysis, Firstly, rigid plant cell walls need to be mechanically disrupted to release the cell content and, in addition to their poor protein yield, plant tissues are rich in proteases and oxidative enzymes, contain phenolic Compounds, starches, oils, pigments and secondary metabolites that massively contaminate protein extracts. In addition, complex matrices of polysaccharides, including large amount of anionic pectins, are present. All these species compete with the binding of proteins to the CPLL beads, impeding proper capture and identification I detection of low-abundance species. When properly pre-treated, plant tissue extracts are amenable to capture by the CPLL beads revealing thus many new species among them low-abundance proteins. Examples are given on the treatment of leaf proteins, of corn seed extracts and of exudate proteins (latex from Hevea brasiliensis). In all cases, the detection of unique gene products via CPLL Capture is at least twice that of control, untreated sample. (c) 2008 Elsevier B.V. All rights reserved.

High-throughput proteomics using matrix-assisted laser desorption/ionization mass spectrometry

It has become evident that the mystery of life will not be deciphered just by decoding its blueprint, the genetic code. In the life and biomedical sciences, research efforts are now shifting from pure gene analysis to the analysis of all biomolecules involved in the machinery of life. One area of these postgenomic research fields is proteomics. Although proteomics, which basically encompasses the analysis of proteins, is not a new concept, it is far from being a research field that can rely on routine and large-scale analyses. At the time the term proteomics was coined, a gold-rush mentality was created, promising vast and quick riches (i.e., solutions to the immensely complex questions of life and disease). Predictably, the reality has been quite different. The complexity of proteomes and the wide variations in the abundances and chemical properties of their constituents has rendered the use of systematic analytical approaches only partially successful, and biologically meaningful results have been slow to arrive. However, to learn more about how cells and, hence, life works, it is essential to understand the proteins and their complex interactions in their native environment. This is why proteomics will be an important part of the biomedical sciences for the foreseeable future. Therefore, any advances in providing the tools that make protein analysis a more routine and large-scale business, ideally using automated and rapid analytical procedures, are highly sought after. This review will provide some basics, thoughts and ideas on the exploitation of matrix-assisted laser desorption/ionization in biological mass spectrometry - one of the most commonly used analytical tools in proteomics - for high-throughput analyses.

Quantitative proteomics using uniform N-15-labeling, MASCOT, and the trans-proteomic pipeline

Stable isotope labeling combined with MS is a powerful method for measuring relative protein abundances, for instance, by differential metabolic labeling of some or all amino acids with N-14 and N-15 in cell culture or hydroponic media. These and most other types of quantitative proteomics experiments using high-throughput technologies, such as LC-MS/MS, generate large amounts of raw MS data. This data needs to be processed efficiently and automatically, from the mass spectrometer to statistically evaluated protein identifications and abundance ratios. This paper describes in detail an approach to the automated analysis of Uniformly N-14/N-15-labeled proteins using MASCOT peptide identification in conjunction with the trans-proteomic pipeline (TPP) and a few scripts to integrate the analysis workflow. Two large proteomic datasets from uniformly labeled Arabidopsis thaliana were used to illustrate the analysis pipeline. The pipeline can be fully automated and uses only common or freely available software.

Mass spectrometry in clinical proteomics - from the present to the future

MS is an important analytical tool in clinical proteomics, primarily in the disease-specific discovery identification and characterisation of proteomic biomarkers and patterns. MS-based proteomics is increasingly used in clinical validation and diagnostic method development. The latter departs from the typical application of MS-based proteomics by exchanging some of the high performance of analysis for the throughput, robustness and simplicity required for clinical diagnostics. Although conventional MS-based proteomics has become an important field in clinical applications, some of the most recent MS technologies have not yet been extensively applied in clinical proteomics. in this review, we will describe the current state of MS in clinical proteomics and look to the future of this field.

Application of semiquantitative proteomics techniques to the Maillard reaction

Proteomic tools-in particular, mass spectrometry (MS)-have advanced significantly in recent years, and the identification of proteins within complex mixtures is now a routine procedure. Quantitative methods of analysis are less well advanced and continue to develop. These include the use of stable isotope ratio approaches, isotopically labeled peptide standards, and nonlabeling methods. This paper summarizes the use of MS as a proteomics tool to identify and semiquantify proteins and their modified forms by using examples of relevance to the Maillard reaction. Finally, some challenges for the future are presented.

The use of proteomics to identify novel therapeutic targets for the treatment of disease

The completion of the Human Genome Project has revealed a multitude of potential avenues for the identification of therapeutic targets. Extensive sequence information enables the identification of novel genes but does not facilitate a thorough understanding of how changes in gene expression control the molecular mechanisms underlying the development and regulation of a cell or the progression of disease. Proteomics encompasses the study of proteins expressed by a population of cells, and evaluates changes in protein expression, post-translational modifications, protein interactions, protein structure and splice variants, all of which are imperative for a complete understanding of protein function within the cell. From the outset, proteomics has been used to compare the protein profiles of cells in healthy and diseased states and as such can be used to identify proteins associated with disease development and progression. These candidate proteins might provide novel targets for new therapeutic agents or aid the development of assays for disease biomarkers. This review provides an overview of the current proteomic techniques available and focuses on their application in the search for novel therapeutic targets for the treatment of disease.

Differential protein expression and subcellular distribution of TGFβ1, β2 and β3 in cardiomyocytes during pressure overload-induced hypertrophy

The transforming growth factorβ(TGFβ) superfamily plays an important role in the myocardial response to hypertrophy. We have investigated the protein expression of TGFβ1,β2andβ3in left ventricular tissue, and determined their subcellular distribution in myocytes by immunoblotting and immunocytochemistry during the development of left ventricular hypertrophy (LVH), using isoform specific antibodies to TGFβ1,β2andβ3. LVH was produced in rats by aortic constriction (AC) and LV tissue was obtained at days (d)0, 1, 3, 7, 14, 21 and 42 following operation. Compared with age matched sham-operated controls (SH), TGFβ1levels in LV tissue of AC rats increased significantly from d1–d14 (P<0.03) concomitant with the adaptive growth of LV tissue. In contrast, TGFβ3levels decreased in LV tissue of AC rats from d3 post-operation (significant from d14–d42,P<0.03). No significant difference in TGFβ2levels were observed from SH and AC rats after operation. Antibodies to TGFβ1stained intercalated disks, sarcolemmal membranes and cytoplasm, but not nuclei, of cardiomyocytes on LV sections from untreated and SH rats. However, a trans-localisation of TGFβ1to the nuclei of cardiomyocytes was observed in AC hearts. Antibodies to TGFβ3stained T tubules, cytoplasm and the nuclei of cardiomyocytes from untreated and SH rats. However, by d7 post-AC operation, TGFβ3expression was lost rapidly from nuclei of cardiomyocytes followed by a reduction in total TGFβ3immunofluorescence in myocytes. Antibodies to TGFβ2stained sarcolemmal membranes of cardiomyocytes from both SH and AC rats without significant difference between groups. Thus, the differential pattern of protein expression and subcellular distribution of TGFβ1,β2andβ3in myocytes during the development of LVH suggests that these molecules play different roles in the response of cardiomyocytes to LVH.

In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria f.sp. hordei

Whilst there is increasing evidence tht the outcome of the interation between a pathogen and a host is dependent on protein-protein interactions, very little information is available on in planta proteomics of biotrophic plant pathogens. Here a proteogenomic approach has been employed to supplement the annotation of the recently sequenced genome and to cast light on the biology of the infection process of the economically important barley powdery mildew pathogen, Blumeria graminis f.sp hordei

MS-based clinical proteomics: biomarker discovery in men’s cancer

Importance of biomarker discovery in men’s cancer diagnosis and prognosis Each year around 10,000 men in the UK die as a result of prostate cancer (PCa) making it the 3rd most common cancer behind lung and breast cancer; worldwide more than 670,000 men are diagnosed every year with the disease [1]. Current methods of diagnosis of PCa mainly rely on the detection of elevated prostate-specific antigen (PSA) levels in serum and/or physical examination by a doctor for the detection of an abnormal prostate. PSA is a glycoprotein produced almost exclusively by the epithelial cells of the prostate gland [2]. Its role is not fully understood, although it is known that it forms part of the ejaculate and its function is to solubilise the sperm to give them the mobility to swim. Raised PSA levels in serum are thought to be due to both an increased production of PSA from the proliferated prostate cells, and a diminished architecture of affected cells, allowing an easier distribution of PSA into the wider circulatory system.