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PubMed2006

[Clinical proteomics in laboratory medicine].

During the past few years, the emphasis on genomics has shifted via transcriptomics to proteomics, the science of understanding how the whole set of proteins are expressed and function at the cellular level. Recent progress in clinical proteomics is mostly thanks to sophisticated new methodologies for proteome analyses. In search for novel biomarkers for cancers and other disorders, we have taken advantage of the surface-enhanced laser/desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) and the MALDI-TOF/TOF. Since personal and verbal reporting of alcohol use is not necessarily accurate, objective markers to assess alcohol consumption are required. We used the ProteinChip SELDI technology to generate comparative protein profiles of the consecutive serum samples obtained during abstinence and found that a 5.9kD peptide detected and identified by this technology could be a novel marker of heavy alcohol consumption. Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We could identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. A number of proteins with altered expression between cancer and adjacent non-cancer tissues were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting and immunohistochemistry. Clinical proteomics consists of the discovery proteomics and the measurement proteomics. Although we still stand at the discovery proteomics, undoubtedly, the clinical proteomics will be a major contributor in laboratory medicine in the near future.

Clinical Laboratory Techniques↗
PubMed2024

Multilevel Proteomics Reveals Epigenetic Signatures in BCG-Mediated Macrophage Activation.

The bacillus Calmette-Guérin BCG vaccine (Mycobacterium bovis) is primarily used to prevent tuberculosis (TB) infections but has wide-ranging immunogenic effects. One of its most notable properties is its ability to induce trained immunity, a memory-like response in innate immune cells such as macrophages. Through targeted analyses of well-established histone marks, prior research has shown that these changes are generated through epigenetic modification. Mass spectrometry-based proteomic approaches provide a way to globally profile various aspects of the proteome, providing data to further identify unexplored mechanisms of BCG-mediated immunomodulation. Here we use multi-level proteomics (total, histone, and phospho to identify networks and potential mechanisms that mediate BCG-induced immunomodulation in macrophages. Histone-focused proteomics and total proteomics were performed at the University of Cape Town (data available via ProteomeXchange with identifier PXD051187), while phosphoproteomics data was retrieved from the ProteomeXchange Repository (identifier PXD013171). We identify several epigenetic mechanisms that may drive BCG-induced training phenotypes. Evidence across the proteomics and histone-focused proteomics data set pair 6 epigenetic effectors (NuA4, NuRD, NSL, Sin3A, SIRT2, SIRT6) and their substrates.

Epigenesis, Genetic↗
PubMed2026

Blood Plasma Analysis in Ovarian Cancer Patients Using an AFM/MS Approach: Effect of Sample Dilution on Proteome Depth.

Early detection of ovarian cancer remains challenging because of the lack of sensitive and reproducible blood-based biomarkers. A major challenge in plasma proteomics is the extremely wide dynamic range of protein concentrations, which prevents simultaneous detection of both high- and low abundance proteins and limits the identification of disease-associated signals. In this study, we applied a combined atomic force microscopy and mass spectrometry (AFM/MS) approach to investigate how sample dilution affects plasma proteome coverage and the detection of differences between healthy donors and patients with stage I and stage III ovarian cancer. Plasma samples were analyzed at two dilution levels (1:100 and 1:10,000). At 1:100 dilution, a total of 235 proteins were identified across all samples, representing the union of all replicates and groups. The reproducible CORE proteome comprised 169 proteins in the Healthy group, 183 in the Stage I group, and 193 in the Stage III group. Differential analysis revealed distinct, non-overlapping protein sets at each dilution level. At 1:100 dilution, most altered proteins were decreased in patients and corresponded to major plasma components, including complement proteins and protease inhibitors. At 1:10,000 dilution, most altered proteins were increased and were predominantly immunoglobulin-related proteins, along with complement regulatory components. These findings show that sample dilution determines which fraction of the plasma proteome is observable. Here, proteome depth refers to the total number of non-redundant proteins accessible within the analytical workflow. When CORE sets from all groups were combined, 216 proteins were identified at 1:100 and 149 at 1:10,000, with 133 shared between the two dilution conditions. The higher dilution contributed 16 additional CORE proteins not detected in the 1:100 CORE union, increasing the combined CORE set to 232 proteins. Thus, higher dilution alone did not increase proteome depth, but provided complementary protein identifications that increased cumulative proteome depth when both dilution conditions were considered together. This effect reflects dilution-dependent selectivity in the composition of the detectable protein subset.

Humans↗
PubMed2003

Thylakoid membrane proteomics.

Proteomics seeks to monitor the global complement of proteins within a cell or organism and accompanying plasticity with respect to development and environment. The proteome is dynamic, the product of current and past gene expression, countless protein-protein interactions and selective proteolytic systems. Consequently the snapshot that a proteomic measurement yields must be integrated into proteome flux; the flow of nutrients and energy through the protein pathways that catalyze and drive life. The thylakoid membrane proteome poses many technical challenges for proteomics. Integral membrane proteins present awkward physico-chemical properties and the abundant photosynthetic machinery conceals much less abundant and no less important proteins such as channels and transporters that control the interaction of stroma and lumen. Discussed here are contrasting approaches to thylakoid proteomics; 'shotgun' techniques that provide throughput benefits by cleaving proteins into smaller more-manageable peptide chunks versus intact protein techniques that provide more detailed and accurate pictures. A two-dimensional chromatography system directly interfaced to electrospray-ionization mass spectrometry has allowed the direct visualization of large reaction-center proteins (up to 83 kDa) from both Photosystems 1 and 2 providing an attractive avenue for characterization of thylakoid membrane proteomes under different conditions because of the ability to resolve molecular heterogeneity resulting from post-translational modifications such as phosphorylation and oxidation. A high-resolution spectrum of Bacteriorhodopsin recorded to an accuracy of 8 ppm using Fourier-transform mass spectrometry demonstrates the first application of this technique to intact polytopic integral membrane proteins.

Journal Article↗
PubMedDate not supplied

Subcellular proteomics.

The step from the analysis of the genome to the analysis of the proteome is not just a matter of numerical complexity in terms of variants of gene products that can arise from a single gene. A significant further level of complexity is introduced by the supramolecular organization of gene products because of protein-protein interactions or targeting of proteins to specific subcellular structures. There is currently no single proteome analysis strategy that can sufficiently address all levels of the organization of the proteome. To approach an appropriate analytical complement for the interrogation of the proteome at all of the levels at which it is organized, there emerges the need for a whole arsenal of proteomics strategies. The proteome analysis at the level of subcellular structures (that can be enriched by subcellular fractionation) represents an analytical strategy that combines classic biochemical fractionation methods and tools for the comprehensive identification of proteins. Among the key potentials of this strategy is the capability to screen not only for previously unknown gene products but also to assign them, along with other known, but poorly characterized gene products, to particular subcellular structures. Furthermore, the analysis at the subcellular level is a prerequisite for the detection of important regulatory events such as protein translocation in comparative studies. This review is meant to give an overview on recent key studies in the field of proteome analysis at the level of subcellular structures, and to highlight potentials and requirements.

Animals↗
PubMedDate not supplied

Applying proteomics technology to platelet research.

Platelets are small enucleated cells that circulate in the blood, where they play a key role in hemostasis and contribute to the formation of vascular plugs. Pathologically, they are involved in thrombosis and heart disease. Because platelets do not have a nucleus, proteomics offers a powerful way to approach their biology. Proteomics technology is based on the huge analytical power offered by mass spectrometry in combination with several separation techniques, such as two-dimensional gel electrophoresis (2DGE) or multidimensional liquid chromatography. Proteomics provides information about the complete set of proteins present in platelets, the platelet proteome, including post-translational variants. Over the last years, several research groups have applied proteomics to platelet research. A detailed analysis of the general proteome and signaling cascades in human platelets will improve our knowledge of platelet function, and thereby aid the development of new therapeutic agents to treat thrombotic diseases. This review presents the major advances in mass spectrometry-based proteomics techniques and their application to platelet research, and analyzes in detail the most relevant contributions to the field.

Biotechnology↗
PubMed2003

Application of proteomics to Pseudomonas aeruginosa.

The recent completion of the Pseudomonas Genome Project, in conjunction with the Pseudomonas Community Annotation Project (PseudoCAP) has fast-tracked our ability to apply the tools encompassed under the term 'proteomics' to this pathogen. Such global approaches will allow the research community to answer long-standing questions regarding the ability of Pseudomonas aeruginosa to survive diverse habitats, its high intrinsic resistance to antibiotics and its pathogenic nature towards humans. Proteomics provides an array of tools capable of confirming the expression of Open Reading Frames (ORF), the relative levels of their expression, the environmental conditions required for this expression and the sub-cellular location of the encoded gene-products. Since proteins are important cellular effectors, the biological questions we pose can be defined in terms of changes in protein expression detectable by separation to purity using two-dimensional gel electrophoresis (2-DGE) and relation to gene sequences via mass spectrometry. As such, we can compare strains with well-characterized phenotypic differences, growth under a variety of stresses, protein interactions and complexes and aid in defining proteins of unknown function. While the complete genome has only recently been finished, a number of studies have already utilized this information and examined various protein gene-products using proteomics. This review summarizes the application of proteomics to P. aeruginosa and highlights potential areas of future research, including overcoming the traditional technical limitations associated with 2-DGE. More focused approaches that target sub-cellular fractions ('sub-proteomes') prior to 2-DGE can provide further functional information. A review of current and previous proteomic projects on P. aeruginosa is presented, as well as theoretical considerations of the importance of sub-proteomic approaches to enhance these investigations.

Amino Acid Sequence↗
PubMed2005

Proteomic analysis of Bacillus anthracis Sterne vegetative cells.

Mass spectrometry and proteomics have found increasing use as tools for the rapid detection of pathogenic bacteria, even when they are in a mixture of non-pathogenic relatives. The success of this technique is greatly augmented by the availability of publicly accessible proteomic databases for specific pathogenic bacteria. To aid proteomic detection analyses for the causative agent of anthrax, we have constructed a comprehensive proteomic catalogue of vegetative Bacillus anthracis Sterne cells using liquid chromatography tandem-mass spectrometry. Proteins were separated by molecular weight or isoelectric point prior to tryptic digestion. Alternatively, the whole protein extract was digested and tryptic peptides were separated by cation exchange chromatography prior to Reverse Phase-LC-MS/MS. The use of three complementary, pre-analytical separation techniques resulted in the identification of 1048 unique proteins, including 694 cytosolic, 153 membrane (including 27 cell wall), and 30 secreted proteins, accounting for 19% of the total predicted proteome. Each identified protein was functionally categorized using the gene attribute database from TIGR CMR. These results provide a large proteomic catalogue of vegetative B. anthracis cells and, coupled with the recent proteomic catalogue of B. anthracis spore proteins, form a thorough summary of proteins expressed in the active and dormant stages of this organism.

Bacillus anthracis↗
PubMed2005

Plastid proteomics.

Plastids are essential organelles present in virtually all cells in plants and in green algae. The proteomes of plastids, and in particular of chloroplasts, have received significant amounts of attention in recent years. Various fractionation and mass spectrometry (MS) techniques have been applied to catalogue the chloroplast proteome and its membrane compartments. Neural network and hidden Markov models, in combination with experimentally derived filters, were used to try to predict the chloroplast subproteomes. Some of the many protein-protein interaction, as well as post-translational modifications have been characterized. Nevertheless, our understanding of the chloroplast proteome and its dynamics is very incomplete. Rapid improvements and wide-scale implementation of MS and new tools for comparative proteomics will undoubtedly accelerate this understanding in the near future. Proteomics studies often generate a large amount of data and these data are only meaningful if they can be easily accessed via the 'world-wide-web' and connected to other types of biological information. The plastid proteome data base (PPDB at http://www.ppdb.tc.cornell.edu/) and other web resources are discussed. This review will briefly summarize recent experimental and theoretical efforts, attempt to translate these data into the functions of the chloroplast and outline expectations and possibilities for (comparative) chloroplast proteomics.

Chloroplasts↗
PubMedDate not supplied

Clinical cancer proteomics: promises and pitfalls.

Proteome analysis promises to be valuable for the identification of tissue and serum biomarkers associated with human malignancies. In addition, proteome technologies offer the opportunity to analyze protein expression profiles and to analyze the activity of signaling pathways. Many published proteomic studies of human tumor tissue are associated with weaknesses in tumor representativity, sample contamination by nontumor cells and serum proteins. Studies often include a moderate number of tumors which may not be representative of clinical materials. It is therefore very important that biomarkers identified by proteomics are validated in representative tumor materials by other techniques, such as immunohistochemistry. Proteome technologies can be used to identify disease markers in human serum. Tumor derived proteins are present at nanomolar to picomolar concentrations in cancer patient sera, 10(6)-10(9)-fold lower than albumin, and will give rise to correspondingly smaller spots/peaks in protein separations. This leads to the need to prefractionate serum samples before analysis. Despite various pitfalls, proteomic analysis is a promising approach to the identification of biomarkers, and for generation of protein expression profiles that can be analyzed by artificial learning methods for improved diagnosis of human malignancy. Recent advances in the field of proteomic analysis of human tumors are summarized in the present review.

Biomarkers, Tumor↗
PubMed2024

Single-tissue proteomics in Caenorhabditis elegans reveals proteins resident in intestinal lysosome-related organelles.

The nematode intestine is the primary site for nutrient uptake and storage as well as the synthesis of biomolecules; lysosome-related organelles known as gut granules are important for many of these functions. Aspects of intestine biology are not well understood, including the export of the nutrients it imports and the molecules it synthesizes, as well as the complete functions and protein content of the gut granules. Here, we report a mass spectrometry (MS)-based proteomic analysis of the intestine of the Caenorhabditis elegans and of its gut granules. Overall, we identified approximately 5,000 proteins each in the intestine and the gonad and showed that most of these proteins can be detected in samples extracted from a single worm, suggesting the feasibility of individual-level genetic analysis using proteomes. Comparing proteomes and published transcriptomes of the intestine and the gonad, we identified proteins that appear to be synthesized in the intestine and then transferred to the gonad. To identify gut granule proteins, we compared the proteome of individual intestines deficient in gut granules to the wild type. The identified gut granule proteome includes proteins known to be exclusively localized to the granules and additional putative gut granule proteins. We selected two of these putative gut granule proteins for validation via immunohistochemistry, and our successful confirmation of both suggests that our strategy was effective in identifying the gut granule proteome. Our results demonstrate the practicability of single-tissue MS-based proteomic analysis in small organisms and in its future utility.

Animals↗
PubMed2005

Peptidyl-Lys metalloendopeptidase-catalyzed 18O labeling for comparative proteomics: application to cytokine/lipolysaccharide-treated human retinal pigment epithelium cell line.

We recently proposed a comparative proteomic method utilizing proteolytic 18O labeling of peptides catalyzed by peptidyl-Lys metalloendopeptidase (Lys-N) (Rao, K. C. S., Carruth, R. T., and Miyagi, M. (2005) Proteolytic 18O labeling by peptidyl-Lys metalloendopeptidase for comparative proteomics. J. Proteome Res. 4, 507-514). Unlike trypsin, which generates a mixture of isotopic isoforms resulting from the incorporation of one or two 18O atoms into each peptide species, Lys-N incorporates only a single 18O atom into the carboxyl terminus of each proteolytically generated peptide in H2(18)O solvent. This study reports the first biological application of the Lys-N-based proteolytic 18O labeling method, characterizing the proteome changes of cytokine/lipopolysaccharide-treated verses untreated human retinal pigment epithelium (ARPE-19) cells. The study resulted not only in the identification of 584 proteins but also the determination of the relative abundances of 562 proteins in the two proteomes. The results demonstrate the usefulness of the Lys-N-based proteolytic 18O labeling method in comparative proteomic studies. The results also provide the most comprehensive description of the retinal pigment epithelium proteome to date.

Amino Acid Sequence↗
PubMed2006

Advances and challenges in liquid chromatography-mass spectrometry-based proteomics profiling for clinical applications.

Recent advances in proteomics technologies provide tremendous opportunities for biomarker-related clinical applications; however, the distinctive characteristics of human biofluids such as the high dynamic range in protein abundances and extreme complexity of the proteomes present tremendous challenges. In this review we summarize recent advances in LC-MS-based proteomics profiling and its applications in clinical proteomics as well as discuss the major challenges associated with implementing these technologies for more effective candidate biomarker discovery. Developments in immunoaffinity depletion and various fractionation approaches in combination with substantial improvements in LC-MS platforms have enabled the plasma proteome to be profiled with considerably greater dynamic range of coverage, allowing many proteins at low ng/ml levels to be confidently identified. Despite these significant advances and efforts, major challenges associated with the dynamic range of measurements and extent of proteome coverage, confidence of peptide/protein identifications, quantitation accuracy, analysis throughput, and the robustness of present instrumentation must be addressed before a proteomics profiling platform suitable for efficient clinical applications can be routinely implemented.

Biomarkers↗
PubMed2004

Chloroplast proteomics: potentials and challenges.

With the available Arabidopsis genome and near-completion of the rice genome sequencing project, large-scale analysis of plant proteins with mass spectrometry has now become possible. Determining the proteome of a cell is a challenging task, which is complicated by proteome dynamics and complexity. The biochemical heterogeneity of proteins constrains the use of standardized analytical procedures and requires demanding techniques for proteome analysis. Several proteome studies of plant cell organelles have been reported, including chloroplasts and mitochondria. Chloroplasts are of particular interest for plant biologists because of their complex biochemical pathways for essential metabolic functions. Information from the chloroplast proteome will therefore provide new insights into pathway compartmentalization and protein sorting. Some approaches for the analysis of the chloroplast proteome and future prospects of plastid proteome research are discussed here.

Chloroplasts↗
PubMed2005

The potential of proteomics and peptidomics for allergy and asthma research.

Progress in the field of proteomics, the branch of biology that studies the full set of proteins derived from a given genome, is moving fast. Two-dimensional gel electrophoresis (2DG) separation of complex protein mixtures and the subsequent analysis of isolated protein spots by mass spectrometry allow fast and accurate identification of proteins. The comparison of spots from different samples separated on customized 2D gels allows the detection of punctual differences in their mobility and facilitates tracing back differences in protein expression, presence of isoforms, splice variants and posttranslational modifications by mass spectrometry. In spite of significant analytical challenges owing to the high complexity of the proteome and the challenge deriving from the necessity to process huge amounts of raw data generated by mass spectrometric profiling, proteomics has evolved to an indispensable tool in life sciences. A restricted window of the proteome that consists of peptides and small proteins not easily manageable by conventional gel electrophoresis prompted the development of separation methods based on liquid chromatography. This new research field termed peptidomics already contributed, together with proteomics to enlarge our knowledge about biological processes and supported by sophisticated bioinformatics tools, to the discovery of new diagnostic and therapeutic targets. The technological capabilities of biophysical separation, mass spectrometry and bioinformatics form the basis of discovery programs that aim at mining the proteome starting from microgram amounts of protein extracts derived from body fluids and tissues. Proteomics and peptidomics have a great potential to speed up allergy and asthma research, where disease- and tissue-specific samples are easy to obtain.

Asthma↗
PubMed2004

Proteomics: current techniques and potential applications to lung disease.

Proteomics aims to study the whole protein content of a biological sample in one set of experiments. Such an approach has the potential value to acquire an understanding of the complex responses of an organism to a stimulus. The large vascular and air space surface area of the lung expose it to a multitude of stimuli that can trigger a variety of responses by many different cell types. This complexity makes the lung a promising, but also challenging, target for proteomics. Important steps made in the last decade have increased the potential value of the results of proteomics studies for the clinical scientist. Advances in protein separation and staining techniques have improved protein identification to include the least abundant proteins. The evolution in mass spectrometry has led to the identification of a large part of the proteins of interest rather than just describing changes in patterns of protein spots. Protein profiling techniques allow the rapid comparison of complex samples and the direct investigation of tissue specimens. In addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. These methodologies have made the application of proteomics on the study of specific diseases or biological processes under clinically relevant conditions possible. The quantity of data that is acquired with these new techniques places new challenges on data processing and analysis. This article provides a brief review of the most promising proteomics methods and some of their applications to pulmonary research.

Animals↗
PubMed2006

Quantitative proteomics reveals posttranslational control as a regulatory factor in primary hematopoietic stem cells.

The proteome is determined by rates of transcription, translation, and protein turnover. Definition of stem cell populations therefore requires a stem cell proteome signature. However, the limit to the number of primary cells available has restricted extensive proteomic analysis. We present a mass spectrometric method using an isobaric covalent modification of peptides for relative quantification (iTRAQ), which was employed to compare the proteomes of approximately 1 million long-term reconstituting hematopoietic stem cells (Lin(-)Sca(+)Kit(+); LSK(+)) and non-long-term reconstituting progenitor cells (Lin(-)Sca(+)Kit(-); LSK(-)), respectively. Extensive 2-dimensional liquid chromatography (LC) peptide separation prior to mass spectrometry (MS) enabled enhanced proteome coverage with relative quantification of 948 proteins. Of the 145 changes in the proteome, 54% were not seen in the transcriptome. Hypoxia-related changes in proteins controlling metabolism and oxidative protection were observed, indicating that LSK(+) cells are adapted for anaerobic environments. This approach can define proteomic changes in primary samples, thereby characterizing the molecular signature of stem cells and their progeny.

Animals↗
PubMed2006

Proteomics in pulmonary medicine.

Proteomics is the study of the entire protein complement of the genome (the proteome) in a biological system. Proteomic studies require a multidisciplinary approach and have only been practical with the convergence of technical and methodologic improvements including the following: advances in mass spectrometry and genomic sequencing that now permit the identification and relative quantization of small amounts (femtomole) of nearly any single protein; new methods in gel electrophoresis that allow the detection of subtle changes in protein expression, including posttranslational modifications; automation and miniaturization that permit high-throughput analysis of clinical samples; and new bioinformatics and computational methods that facilitate analysis and interpretation of the abundant data that are generated by proteomics experiments. This convergence makes proteomics studies practical for pulmonary researchers using BAL fluid, lung tissue, blood, and exhaled breath condensates, and will facilitate the research of complex, multifactorial lung diseases such as acute lung injury and COPD. This review describes how proteomics experiments are conducted and interpreted, their limitations, and how proteomics has been used in clinical pulmonary medicine.

Humans↗