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PubMed2006

Direct-tissue SELDI-TOF mass spectrometry analysis: a new application for clinical proteomics.

BACKGROUND: New molecular profiling technologies can aid in analysis of small pathologic samples obtained by minimally invasive biopsy and may enable the discovery of key biomarkers synergistic with anatomopathologic analysis related to prognosis, therapeutic response, and innovative target validation. Thus proteomic analysis at the histologic level in healthy and pathologic settings is a major issue in the field of clinical proteomics. METHODS: We used surface-enhanced laser desorption ionization-time-of-flight mass spectrometry (SELDI-TOF MS) technology with surface chromatographic subproteome enrichment and preservation of the spatial distribution of proteomic patterns to detect discrete modifications of protein expression. We performed in situ proteomic profiling of mouse tissue and samples of human cancer tissue, including brain and lung cancer. RESULTS: This approach permitted the discrimination of glioblastomas from oligodendrogliomas and led to the identification of 3 potential markers. CONCLUSION: Direct tissue proteomic analysis is an original application of SELDI-TOF MS technology that can expand the use of clinical proteomics as a complement to the anatomopathological diagnosis.

Animals↗
PubMed2003

Proteomics as a functional genomics tool.

To understand the function of all the genes in an organism, one needs to know not only which genes are expressed, when, and where, but also what the protein end products are and under which conditions they accumulate in certain tissues. Proteomics aims at describing the whole protein output of the genome and complements transcriptomic and metabolomic studies. Proteomics depends on extracting, separating, visualizing, identifying, and quantifying the proteins and their interactions present in an organism or tissue at any one time. All of these stages have limitations. Therefore, it is, at present, impossible to describe the whole proteome of any organism. Plants might synthesize many thousands of proteins at one time, and the whole potentially synthesized proteome certainly exceeds the number of estimated genes for that genome. This occurs because the gene products of one gene can differ due to alternative splicing and a variety of possible posttranslational modifications. It is, therefore, essential to optimize every step towards detecting the whole proteome while realizing the limitations. We concentrate here on the most commonly used steps in high-throughput plant proteomics with the techniques we have found most reproducible and with the highest resolution and quality.

Coloring Agents↗
PubMed2006

Proteomics with two-dimensional gel electrophoresis and mass spectrometry analysis in cardiovascular research.

Proteomics is a large-scale, comprehensive study of the proteins of a cell or organism. It is a unique means of characterizing proteins that are expressed in a cell or tissue at any given time-point and of identifying any modifications that they may undergo. Thus, it is a powerful technology that can detect and identify the changes of the structure and function of proteins in response to intra- and extracellular environmental signals or disease states. As proteomics can establish a link for genes and proteins with a disease, it will play an important role in defining the molecular determinants of a disease and in identifying targets for drug discoveries and diagnostics. We have carried out the first proteomics study for coronary artery disease (CAD) and found that the expression of the ferritin light chain was significantly increased in CAD tissues. In this chapter, we use the CAD study as an example to demonstrate the procedures involved in proteomics analysis. The proteome is visualized by two-dimensional gel electrophoresis, a powerful and widely used method for proteomics, and the proteins of interest are then identified by mass spectrometry. This technique should be useful in characterizing cardiovascular diseases and in defining signaling pathways for cardiovascular development and physiology.

Cardiovascular Diseases↗
PubMed2002

Current perspectives in cancer proteomics.

Proteome technology has been used widely in cancer research and is a useful tool for the identification of new cancer markers and treatment-related changes in cancer. This article details the use of proteome technology in cancer research, and laboratory-based and clinical cancer research studies are described. New developments in proteome technology that enable higher sample-throughput are evaluated and methods for enhancing conventional proteome analysis (based on two-dimensional electrophoresis) discussed. The need to couple laboratory-based proteomics research with clinically relevant models of the disease is also considered, as this remains the next main challenge of cancer-related proteome research.

Humans↗
PubMed2000

Applications of proteomics in oncology.

Genomics has expanded the field of molecular oncology, and proteomics is complementing genomics in the fields of elucidation of pathophysiology, gene function, molecular diagnosis and anticancer drug discovery. This trend is reflected in the establishment of the Human Tumour Gene Index by the National Cancer Institute (NCI), which is now followed by the Tissue Proteomics Initiative. Laser capture microdissection (LCM) provides an ideal method for extraction of cells from specimens in which the exact morphologies of both the captured cells and the surrounding tissue are preserved. Proteomic technologies can be applied for the further characterisation and analysis of proteins. LCM can also be combined with the protein chip technology. Proteomic technologies have been used for the study of cancer of various organs including the liver, prostate, breast, bladder and oesophagus. Some of the anticancer strategies are directed against proteases that facilitate several steps in cancer progression. Proteomic mapping of blood vessels in normal and malignant tissues can be used to identify tissue-specific markers on the endothelium that serve as potential targets for in vivo drug delivery. Studies of global protein expression in human tumours have led to the identification of various polypeptide markers, potentially useful as diagnostic tools. Genes that encode proteins that are overexpressed in tumours are being identified. Demonstration of tissue or cell type specific expression of some nuclear matrix proteins has led to the search for tumour specific nuclear matrix proteins. There is considerable activity in the commercial sector to develop diagnostic tests, as well as to facilitate anticancer drug discovery using proteomic technologies. Continued refinement of techniques and methodologies to determine the abundance and status of proteins in vivo holds great promise for future study of normal cells and associated neoplasms.

Animals↗
PubMed2002

Functional proteomics to study protection of the ischaemic myocardium.

Mechanisms to reduce the deleterious effects of myocardial ischaemia are of particular clinical importance and have been the focus of intense research for a number of years. Among novel approaches to studying the ischaemic heart, proteomics, or the analysis of all cellular proteins, presents as a powerful method to deconstruct the mechanisms of disease and protection. Specifically, the field of functional proteomics is an emerging application of proteomics that melds aspects of classical proteomics, biochemistry, molecular biology and physiology into an approach that facilitates an understanding of how proteins and protein interactions engender phenotype. This review highlights different types of proteomic applications and provides a prospectus for functional proteomics as a robust vehicle driving drug discovery and design.

Animals↗
PubMed2004

High-throughput proteomics using antibody microarrays.

Antibody-based microarrays are a novel technology that hold great promise in proteomics. Microarrays can be printed with thousands of recombinant antibodies carrying the desired specificities, the biologic sample (e.g., an entire proteome) and any specifically bound analytes detected. The microarray patterns that are generated can then be converted into proteomic maps, or molecular fingerprints, revealing the composition of the proteome. Using this tool, global proteome analysis and protein expression profiling will thus provide new opportunities for biomarker discovery, drug target identification and disease diagnostics, as well as providing insights into disease biology. Intense work is currently underway to develop this novel technology platform into the high-throughput proteomic tool required by the research community.

Antibodies↗
PubMed2005

Proteomics in cancer vaccine development.

Proteomics is a new scientific field aimed at the large-scale characterization of the protein constituents of biologic systems. It facilitates comparisons between different protein preparations by searching for minute differences in their protein expression repertoires and the patterns of their post-translational modifications. These attributes make proteomics perfectly suited for searching for proteins and peptides expressed exclusively or preferentially in cancer cells as candidates for cancer vaccines. The main proteomics technologies include 2D polyacrylamide gel electrophoresis, multidimensional high-performance liquid chromatography, mass spectrometry and protein arrays. Proteomics technologies used to analyze cancer culture cells, fresh tumor specimens, human leukocyte antigen peptides, serum and serum antibodies (serologic proteomics) have successfully identified tumor markers. Turning the potential vaccine candidates identified by proteomics technologies into clinical treatments awaits demonstration.

Animals↗
PubMed2005

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.

Computational Biology↗
PubMed2005

Gel-based versus gel-free proteomics: a review.

With the sequencing of the genome of over 150 organisms, the field of biology has been revolutionised. Instead of studying one gene or protein at the time, it is now possible to study the effect of physiological or pathological changes on the expression of all genes or proteins in the organism. Proteomics aims at the simultaneous analysis of all proteins expressed by a cell, tissue or organism in a specific physiological condition. Because proteins are the effector molecules in all organisms, it is evident that changes in the physiological condition of an organism will be reflected by changes in protein expression and/or processing. Since the formulation of the concept of proteomics in the mid 90's proteomics has relied heavily on 2 dimensional gel electrophoresis (2DGE) for the separation and visualization of proteins. 2DGE, however, has a number of inherent drawbacks. 2DGE is costly, fairly insensitive to low copy proteins and cannot be used for the entire proteome. Therefore, over the years, several gel-free proteomics techniques have been developed to either fill the gaps left by 2DGE or to entirely abolish the gel based techniques. This review summarizes the most important gel-free and gel-based proteomics techniques and compares their advantages and drawbacks.

Electrophoresis, Polyacrylamide Gel↗
PubMed2002

Proteomics and a future generation of plant molecular biologists.

Proteomic methods are required for the study of many different aspects of plant function. Important issues in proteomics include the molecular complexity of proteins, given that there are hundreds of thousands of chemically and physically distinct proteins in plants, and the context of protein functions with respect to both genomes and the environment. Available genomic and gene sequences greatly simplify the identification of proteins using improved techniques of mass spectrometry. This improved capability has led to much discussion on proteomes, and some experimentation using proteomic methodologies aimed at modest numbers of proteins. The scale of proteomics is open, for the number of proteins and genes considered at any one time is as dependent on the nature of the scientific question posed as on technical resources and capabilities. We know just enough about plant proteomes to imagine the breathtaking scope of our ignorance. There are tremendous opportunities for new molecular biologists to define the nature of the protein machines that transduce genetic and environmental information, and transform simple energy and matter, to give plants.

Amino Acid Sequence↗
PubMed2003

Affordable proteomics: the two-hybrid systems.

Numerous proteomic methodologies exist, but most require a heavy investment in expertise and technology. This puts these approaches out of reach for many laboratories and small companies, rarely allowing proteomics to be used as a pilot approach for biomarker or target identification. Two proteomic approaches, 2D gel electrophoresis and the two-hybrid systems, are currently available to most researchers. The two-hybrid systems, though accommodating to large-scale experiments, were originally designed as practical screens, that by comparison to current proteomics tools were small-scale, affordable and technically feasible. The screens rapidly generated data, identifying protein interactions that were previously uncharacterized. The foundation for a two-hybrid proteomic investigation can be purchased as separate kits from a number of companies. The true power of the technique lies not in its affordability, but rather in its portability. The two-hybrid system puts proteomics back into laboratories where the output of the screens can be evaluated by researchers with experience in the particular fields of basic research, cancer biology, toxicology or drug development.

Gene Library↗
PubMed2003

Mapping the proteome of Leishmania Viannia parasites using two-dimensional polyacrylamide gel electrophoresis and associated technologies.

In this study we have demonstrated the potential of two-dimensional electrophoresis (2DE)-based technologies as tools for characterization of the Leishmania proteome (the expressed protein complement of the genome). Standardized neutral range (pH 5-7) proteome maps of Leishmania (Viannia) guyanensis and Leishmania (Viannia) panamensis promastigotes were reproducibly generated by 2DE of soluble parasite extracts, which were prepared using lysis buffer containing urea and nonidet P-40 detergent. The Coomassie blue and silver nitrate staining systems both yielded good resolution and representation of protein spots, enabling the detection of approximately 800 and 1,500 distinct proteins, respectively. Several reference protein spots common to the proteomes of all parasite species/strains studied were isolated and identified by peptide mass spectrometry (LC-ES-MS/MS), and bioinformatics approaches as members of the heat shock protein family, ribosomal protein S12, kinetoplast membrane protein 11 and a hypothetical Leishmania-specific 13 kDa protein of unknown function. Immunoblotting of Leishmania protein maps using a monoclonal antibody resulted in the specific detection of the 81.4 kDa and 77.5 kDa subunits of paraflagellar rod proteins 1 and 2, respectively. Moreover, differences in protein expression profiles between distinct parasite clones were reproducibly detected through comparative proteome analyses of paired maps using image analysis software. These data illustrate the resolving power of 2DE-based proteome analysis. The production and basic characterization of good quality Leishmania proteome maps provides an essential first step towards comparative protein expression studies aimed at identifying the molecular determinants of parasite drug resistance and virulence, as well as discovering new drug and vaccine targets.

Animals↗
PubMed2026

Mapping the covalent cysteine interactome of Ebselen reveals high-sensitivity target engagement and redox proteome remodeling.

Ebselen is a covalent organoselenium compound with broad pharmacological activity, yet its cellular cysteine targets and downstream proteomic consequences remain incompletely defined. Here, we integrated competitive gel-based activity-based protein profiling, reactivity-dependent tandem orthogonal proteolysis-activity-based protein profiling, and TMT-based quantitative proteomics to map Ebselen-induced cysteine engagement and proteome remodeling in living cancer cells. Ebselen exhibited dose-dependent cytotoxicity and markedly perturbed intracellular thiol-redox balance, as reflected by glutathione depletion and altered reactive oxygen species-associated fluorescence readouts. Competitive gel-based profiling confirmed concentration-dependent engagement of protein cysteine residues in live cells. Quantitative rdTOP-ABPP further identified hundreds of dose-responsive cysteine sites in HeLa and HepG2 cells and revealed a preference for cysteine microenvironments enriched with basic residues. Cross-cell-line comparison highlighted CDK5 Cys53, SMU1 Cys298, and RPSA2 Cys163 as conserved covalent nodes, among which CDK5 Cys53 showed high sensitivity to Ebselen treatment, a finding validated by competitive labeling and MS-based site assignment. Global TMT proteomics revealed extensive remodeling of redox-related and cell-survival-associated pathways, including compensatory upregulation of selenoproteins such as TXNRD1 and GPX family members. Together, these results define a chemical proteomic atlas of Ebselen-cysteine interactions and provide a framework for understanding and optimizing covalent organoselenium therapeutics.

Humans↗
PubMed2026

Saliva and salivary pellicle composition and proteomic profile in smokers vs. non-smokers and its effect on dental erosion.

OBJECTIVE: To analyse the salivary composition and proteomic profile of saliva and the salivary pellicle in smokers compared to non-smokers, and to examine potential differences in the erosion-protective capacity of the salivary pellicle. METHODS: Twenty-five smokers and 25 non-smokers were included. Unstimulated and stimulated saliva samples were analysed regarding flow rate, pH, buffer capacity, calcium, phosphate, fluoride, and protein content. Saliva and salivary pellicle samples were analysed by data-independent acquisition mass spectrometry (DIA-MS) for proteome profiling. In an in situ experiment, intraoral splints were loaded with bovine enamel and dentine specimens for 120 min. Pellicle-covered specimens were extraorally eroded (HCl, pH 2.3, 60 s). Calcium release was determined photometrically and compared to pellicle-free controls. RESULTS: Except for phosphate in stimulated saliva (padj.=0.003), salivary parameters were not significantly different between smokers and non-smokers. Proteome profiling detected 1759±154 proteins (cumulative 1963) in saliva, and 4262±362 proteins (cumulative 4625) in the salivary pellicle. The relative abundances of 282 (unstimulated saliva), 338 (stimulated saliva), and 4 (salivary pellicle) protein groups differed significantly between smokers and non-smokers. Functional enrichment analysis of differentially abundant human proteins revealed biological processes such as coagulation, immune response, and carcinogenic reactive oxygen species processes to be impacted by smoking. The salivary pellicle had a significant erosion-protective effect in enamel compared to the control (41.4 ± 6.3 nmol/mm2), but no differences between smokers (33.2 ± 10.6 nmol/mm2, padj.=0.001) and non-smokers (32.7 ± 8.6 nmol/mm2, padj.=0.001) were found. CONCLUSION: The proteomic profiles of both unstimulated and stimulated saliva and the salivary pellicle differ between smokers and non-smokers. CLINICAL SIGNIFICANCE: Despite the different proteomic profiles indicating a significant impact of smoking on the oral cavity, the erosion-protective capacity of the salivary pellicle of smokers and non-smokers does not differ.

Dental Pellicle↗
PubMed2026

Plasma proteomic profiling characterizes candidate biomarkers of perimesencephalic non-aneurysmal subarachnoid hemorrhage.

OBJECT: This study aims to explore the plasma proteomic profiles of angiographically confirmed pmSAH and aSAH, and to identify candidate protein biomarkers for discriminating these subtypes on a biological level. METHODS: The differentially abundant proteins of plasma samples from patients with pmSAH (n = 30) and aSAH (n = 30) were analyzed by data-independent acquisition proteomics, and candidate biomarkers were screened. RESULTS: 291 candidate biomarkers were obtained that could be used to distinguish pmSAH patients from aSAH patients, among which 76 were upregulated and 215 were downregulated in pmSAH. Subsequently, the 10 candidate biomarkers were validated by enzyme-linked immunosorbent assay in a validation cohort of 72 subjects. ORM1, ORM2, HP and NMNAT1 were specifically down-regulated in the pmSAH group, while ANP32A was specifically up-regulated in the pmSAH group. FGL2 was specifically up-regulated in the aSAH group. The combined model of ORM2, HP and ANP32A had the best discriminative power (AUC = 0.880). CONCLUSIONS: This study identified ORM2, HP, and ANP32A as candidate biomarkers reflecting biological differences between pmSAH and aSAH. SIGNIFICANCE: Although some proteomic studies have analyzed aneurysmal subarachnoid hemorrhage, to date, there have been no reports on the circulating proteomic analysis of pmSAH. Comparative analysis of the circulating proteomic differences between pmSAH and aSAH may not only help understand the causes of pmSAH, but also contribute to a deeper understanding of mechanisms showing how pmSAH differs from the formation and rupture mechanisms of intracranial aneurysms.

Humans↗
PubMed2026

Proteome-wide Ubiquitinome Profiling Reveals Substrate-specific Dynamics Within the USP7 Network.

USP7 is a pleiotropic deubiquitylating enzyme that is involved in tumor suppression, (neuro) development, chromatin regulation and the DNA damage response. How USP7 regulates these diverse pathways is still unclear. Here, we report data-independent acquisition and label free quantitation mass spectrometry to profile the proteome-wide impact of USP7 on substrate de-ubiquitylation and overall protein abundance. First, we identified proteins associated with endogenous USP7 by immunopurification followed by data-independent acquisition and label free quantitation mass spectrometry. Integration of our new results with earlier interactomes of epitope-tagged USP7 yielded a consensus set of high-confidence protein targets. Domain mapping analysis revealed that, in addition to the TRAF domain, the ubiquitin-like domains of USP7 play a key role in substrate selection. Using specific enrichment of tryptic K-ε-GG peptides, we mapped proteome-wide changes in ubiquitinome dynamics following inhibition of USP7. Combining unbiased proteome-wide and targeted quantitative mass spectrometry revealed that deubiquitylation by USP7 can have different effects on the stability of distinct substrates, and suggests that USP7's activity profile is substrate-dependent rather than an intrinsic enzymatic property. Thus, in addition to providing a proteome-wide map of USP7 target sites, our multi-angle proteomics approach reveals that the effects of USP7-mediated deubiquitylation on its targets are remarkably variable and substrate-specific. Finally, based on these detailed molecular insights we show how USP7 connects various neurodevelopmental syndromes and tumor suppression pathways.

Ubiquitin-Specific Peptidase 7↗
PubMed2026

Spatial Proteomics of the Normal Breast Collagen Stroma: Links to Density and Body Mass Index.

Collagen breast stroma can become a breast cancer risk factor, yet proteomic regulation of normal breast stroma remains poorly defined. This study evaluates the spatial regulation of the collagen proteome from normal breast tissue. Normal breast tissue sections from the Susan G. Komen tissue bank were used (n = 40), with data including genetic ancestry (n = 20 African ancestry; n = 20 European ancestry), body-mass-index (BMI), age, and mammogram density by the Breast Imaging Reporting and Data System (BI-RADS). 10-plex cell marker staining showed CD44 and COL1A1 markers modulated with BMI. Collagen fiber widths by second harmonic generation microscopy contrasted in BMI categories by genetic ancestry. Targeted extracellular matrix proteomics mass spectrometry imaging showed the collagen alpha-1(I) chain proteome was spatially heterogeneous across the normal breast microenvironment with site-specific post-translational modification of proline hydroxylation. Signatures computationally extracted from stroma-rich regions reported that 47 collagen peptides distinguished BI-RADS categories (area under the receiver operating curve >0.7; p-value >0.05). Multivariate modeling of collagen peptides, fiber metrics, and clinical features supported a strong positive association with BMI as a determinant of collagen alterations in the normal breast. This study provides a foundation for larger studies investigating the clinical value of spatial collagen proteome alterations in human breast.

Humans↗