Use Of Proteomics Research Articles

Mass Spectrometry and the Evolution of Proteomics

Mass Spectrometry and the Evolution of Proteomics

Towards proteome standards: The use of absolute quantitation in high-throughput biomarker discovery

The use of proteomics to profile biological fluids and identify therein biomarkers for cancer and other diseases was initially received with considerable excitement. However, results have fallen short of the expectations. Traditionally, protein biomarkers have been identified by measurement of relative expression changes between case and control samples from which differentially expressed proteins are then considered to represent biomarker candidates. We argue that current individual proteomics-based biomarker discovery studies lack the statistical strength for the identification of high-confidence biomarkers. Instead, multi-group efforts are necessary to facilitate the generation of sufficient sample sizes. This is contingent on the ability to collate and cross-compare data from different studies, which will require the use of a common metric or standards. Though profound, the technical challenges for absolute protein quantification can be overcome. The use of matrix specific, shared standards for absolute quantitation presents an opportunity to facilitate the much needed, but currently impossible, comparisons of different studies. In addition to community-wide approaches to standardize pre-analytical biomarker research studies, it is also important to establish means to integrate experimental data from different studies in order to assess the usefulness of proposed biomarkers with sufficient statistical certainty.

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.

Use of proteomics to identify highly abundant maternal factors that drive the egg-to-embryo transition

As IVF becomes an increasingly popular method for human reproduction, it is more critical than ever to understand the unique molecular composition of the mammalian oocyte. DNA microarray studies have successfully provided valuable information regarding the identity and dynamics of factors at the transcriptional level. However, the oocyte transcribes and stores a large amount of material that plays no obvious role in oogenesis, but instead is required to regulate embryogenesis. Therefore, an accurate picture of the functional state of the oocyte requires both transcriptional profiling and proteomics. Here, we summarize our previous studies of the oocyte proteome, and present new panels of oocyte proteins that we recently identified in screens of metaphase II-arrested mouse oocytes. Importantly, our studies indicate that several abundant oocyte proteins are not, as one might predict, ubiquitous housekeeping proteins, but instead are unique to the oocyte. Furthermore, mouse studies indicate that a number of these factors arise from maternal effect genes (MEGs). One of the identified MEG proteins, peptidylarginine deiminase 6, localizes to and is required for the formation of a poorly characterized, highly abundant cytoplasmic structure: the oocyte cytoplasmic lattices. Additionally, a number of other MEG-derived abundant proteins identified in our proteomic screens have been found by others to localize to another unique oocyte feature: the subcortical maternal complex. Based on these observations, we put forth the hypothesis that the mammalian oocyte contains several unique storage structures, which we have named maternal effect structures, that facilitate the oocyte-to-embryo transition.

Tubers from potato lines expressing a tomato Kunitz protease inhibitor are substantially equivalent to parental and transgenic controls

Recombinant protease inhibitors represent useful tools for the development of insect-resistant transgenic crops, but questions have been raised in recent years about the impact of these proteins on endogenous proteases and chemical composition of derived food products. In this study, we performed a detailed compositional analysis of tubers from potato lines expressing the broad-spectrum inhibitor of Ser and Asp proteases, tomato cathepsin D inhibitor (SlCDI), to detect possible unintended effects on tuber composition. A compositional analysis of key nutrients and toxic chemicals was carried out with tubers of SlCDI-expressing and control (comparator) lines, followed by a two-dimensional gel electrophoresis (2-DE) proteomic profiling of total and allergenic proteins to detect eventual effects at the proteome level. No significant differences were observed among control and SlCDI-expressing lines for most chemicals assayed, in line with the very low abundance of SlCDI in tubers. Likewise, proteins detected after 2-DE showed no quantitative variation among the lines, except for a few proteins in some control and test lines, independent of slcdi transgene expression. Components of the patatin storage protein complex and Kunitz protease inhibitors immunodetected after 2-DE showed unaltered deposition patterns in SlCDI-expressing lines, clearly suggesting a null impact of slcdi on the intrinsic allergenic potential of potato tubers. These data suggest, overall, a null impact of slcdi expression on tuber composition and substantial equivalence between comparator and SlCDI-expressing tubers despite reported effects on leaf protein catabolism. They also illustrate the usefulness of proteomics as a tool to assess the authenticity of foods derived from novel-generation transgenic plants.

Use of proteomics in radiobiological research: current state of the art

Biological systems are complex, variable and to a great extent adaptive to environmental and occupational challenge such as ionising radiation, making the mathematical modelling of their behaviour a diYcult task. The required models need to be based on useful experimental data describing global eVects on a cellular, tissue and organ level. High-throughput technologies such as proteomics have been shown to be powerful tools on many areas of modern biology. In radiation biology, especially facing the question of possible adverse health eVects following exposures to low doses of ionising and non-ionising radiation, new and sensitive approaches are slowly gaining support. Epidemiological studies suggest that doses of ionising radiation much lower than previously assumed may cause adverse eVects on human health. However, the epidemio

What is stemness?

This paper, addressed to both philosophers of science and stem cell biologists, aims to reduce the obscurity of and disagreements over the nature of stemness. The two most prominent current theories of stemness—the entity theory and the state theory—are both biologically and philosophically unsatisfactory. Improved versions of these theories are likely to converge. Philosophers of science can perform a much needed service in clarifying and formulating ways of testing entity and state theories of stemness. To do so, however, philosophers should acquaint themselves with the latest techniques and approaches employed by bench scientists, such as the use of proteomics, genome-wide association studies, and ChIP-on-chip arrays. An overarching theme of this paper is the desirability of bringing closer together the philosophy of science and the practice of scientific research.

Proteomics Analysis of Human Skeletal Muscle Reveals Novel Abnormalities in Obesity and Type 2 Diabetes

OBJECTIVEInsulin resistance in skeletal muscle is an early phenomenon in the pathogenesis of type 2 diabetes. Studies of insulin resistance usually are highly focused. However, approaches that give a more global picture of abnormalities in insulin resistance are useful in pointing out new directions for research. In previous studies, gene expression analyses show a coordinated pattern of reduction in nuclear-encoded mitochondrial gene expression in insulin resistance. However, changes in mRNA levels may not predict changes in protein abundance. An approach to identify global protein abundance changes involving the use of proteomics was used here.RESEARCH DESIGN AND METHODSMuscle biopsies were obtained basally from lean, obese, and type 2 diabetic volunteers (n = 8 each); glucose clamps were used to assess insulin sensitivity. Muscle protein was subjected to mass spectrometry–based quantification using normalized spectral abundance factors.RESULTSOf 1,218 proteins assigned, 400 were present in at least half of all subjects. Of these, 92 were altered by a factor of 2 in insulin resistance, and of those, 15 were significantly increased or decreased by ANOVA (P < 0.05). Analysis of protein sets revealed patterns of decreased abundance in mitochondrial proteins and altered abundance of proteins involved with cytoskeletal structure (desmin and alpha actinin-2 both decreased), chaperone function (TCP-1 subunits increased), and proteasome subunits (increased).CONCLUSIONSThe results confirm the reduction in mitochondrial proteins in insulin-resistant muscle and suggest that changes in muscle structure, protein degradation, and folding also characterize insulin resistance.

Use of proteomics for validation of the isolation process of clotting factor IX from human plasma

The use of proteomic techniques in the monitoring of different production steps of plasma-derived clotting factor IX (pd F IX) was demonstrated. The first step, solid-phase extraction with a weak anion-exchange resin, fractionates the bulk of human serum albumin (HSA), immunoglobulin G, and other non-binding proteins from F IX. The proteins that strongly bind to the anion-exchange resin are eluted by higher salt concentrations. In the second step, anion-exchange chromatography, residual HSA, some proteases and other contaminating proteins are separated. In the last chromatographic step, affinity chromatography with immobilized heparin, the majority of the residual impurities are removed. However, some contaminating proteins still remain in the eluate from the affinity column. The next step in the production process, virus filtration, is also an efficient step for the removal of residual impurities, mainly high molecular weight proteins, such as vitronectin and inter-alpha inhibitor proteins. In each production step, the active component, pd F IX and contaminating proteins are monitored by biochemical and immunochemical methods and by LC-MS/MS and their removal documented. Our methodology is very helpful for further process optimization, rapid identification of target proteins with relatively low abundance, and for the design of subsequent steps for their removal or purification.

Blood-related proteomics

Blood-related proteomics is an emerging field, recently gaining momentum. Indeed, a wealth of data is now available and a plethora of groups has contributed to add pieces to the jigsaw puzzle of protein complexity within plasma and blood cells. In this review article we purported to sail across the mare magnum of the actual knowledge in this research endeavour. The main strides in proteomic investigations on red blood cells, platelets, plasma and white blood cells are hereby presented in a chronological order. Moreover, a glance is given at prospective studies which promise to shift the focus of attention from the end product to its provider, the donor, in a sort of Kantian "Copernican revolution". A well-rounded portrait of the usefulness of proteomics in blood-related research is accurately given. In particular, proteomic tools could be adopted to follow the main steps of the blood-banking production processes (a comparison of collection methods, pathogen inactivation techniques, storage protocols). Thus proteomics has been recently transformed from a mere basic-research extremely-expensive toy into a dramatically-sensitive and efficient eye-lens to either delve into the depths of the molecular mechanisms of blood and blood components or to establish quality parameters in the blood-banking production chain totally anew.

Perspective: Proteomic approach to detect biomarkers of human growth hormone

Several serum biomarkers for recombinant human growth hormone (rhGH) have been established, however, none alone or in combination have generate a specific, sensitive, and reproducible ‘kit’ for the detection of rhGH abuse. Thus, the search for additional GH specific biomarkers continues. In this review, we focus on the use of proteomics in general and two-dimensional electrophoresis (2-DE) in particular for the discovery of new GH induced serum biomarkers. Also, we review some of the protocols involved in 2-DE. Finally, the possibility of tissues other than blood for biomarker discovery is discussed.

Proteomics: addressing the challenges of osteoarthritis

Osteoarthritis (OA) is the most frequent rheumatic disease and is the leading cause of disability in developed countries. Its poorly understood pathophysiology limits the discovery of targets for pharmacological intervention and there are few effective medical treatments beyond pain control and surgery. Proteomic technologies may help identifying new targets of OA as well as diagnostic or therapeutic biomarkers, which has stimulated interest in this field. In this review, we discuss the most recent findings arising from the use of proteomics for the identification of OA biomarkers in synovial fluid and serum, and for the discovery of possible therapeutic targets in cartilage or by using chondrocyte culture systems.

Anatomic site‐specific proteomic signatures of gastrointestinal stromal tumors

AbstractThe gastrointestinal stromal tumor (GIST) is the most common mesenchymal malignancy of the gastrointestinal tract. Its clinical course ranges widely from a curable disorder to a highly malignant disease. Although its clinical and molecular characteristics depend on the anatomic site of origin, the molecular background of GIST arising in different anatomical site has not been studied yet. To investigate the proteomic background of GIST, we examined the proteomic features corresponding to the anatomic site of tumor origin. Comparison of the proteomic profile of gastric (23 cases) and small intestinal (9 cases) GIST by 2‐DE revealed 105 protein spots with significantly different intensity (p &lt;0.01) between the two groups. Mass spectrometric study identified 68 distinct proteins for these 105 protein spots, including cancer‐associated ones such as prohibitin, pigment epithelium‐derived factor, and alpha‐actinin 4. The intensity of 37/105 (35.2%) protein spots was significantly concordant with the corresponding mRNA levels (p &lt;0.01). Although both 2‐D DIGE and microarray experiments showed significant up‐regulation of vimentin expression in small intestinal GIST, Western blotting did not show a significant difference between the two groups. In conclusion, our study demonstrates the proteins specially expressed in GIST depending on their site of origin, as well as the unique advantage offered by use of proteomics to acquire such data. The identified proteins may provide clues to understanding the different characteristics of GIST depending on their site of origin.

Use of proteomics for design of a tailored host cell for highly efficient protein purification

After some initial optimization, a downstream process comprised of one or several chromatography steps removes the majority of the host proteins and achieves a reasonable degree of purification. The separation of remaining contaminant proteins from the target protein could become very difficult and costly due to their similar physicochemical properties. In this paper we describe a highly efficient strategy, based on proteomic analysis and elution chromatography, by which a protein of interest may be isolated from copurifying contaminants. Mutant strains of Escherichia coli were prepared that are deficient in three prevalent host proteins found in a strategic fraction of an elution profile of nickel immobilized affinity chromatography. Recombinant green fluorescent protein (GFPuv) served as a model protein and its elution was directed to this optimized fraction with an N-terminus hexahistidine tag (his 6), thereby easing its recovery. We demonstrate that proteomic data can facilitate the rational engineering of host cell expressing the target protein and the design of an efficient process for its purification.

Gel-based proteomics approach for detecting low nitrogen-responsive proteins in cultivated rice species

Nitrogen fertilization is essential for increasing rice production to meet the food demands of increasing world's population. We established an in vivo hydroponic rice seedling culture system to investigate physio-biochemical/molecular responses of various rice japonica and indica cultivars to low nitrogen (N). Three-week-old seedlings grown in Yoshida's nutrient solution manifested stable and reproducible symptoms, such as reduced shoot growth and length under low N. Out of 12 genetically selected cultivars, 11 cultivars showed varied degrees of growth reduction response to applied N (4 and 40 ppm N for treatment and control, respectively), whereas one cultivar (no. 12) showed similar growth as the control though its leaf width was smaller than control. Leaves of a representative low N-responsive cultivar (BG90-2) were sampled for revealing protein profiles between low and normal (control) N application by two-dimensional gel electrophoresis (2-DGE). Forty-one proteins were identified with MALDI-TOF-MS and nESI-LC-MS/MS. Assignment of proteins into major (energy metabolism, photosynthesis and oxidative stress) and minor functional categories, revealed many novel low N-responsive proteins, including those having energy/photosynthesis- and defense/stress- and iron homeostasis-related functions. Results suggest the usefulness of proteomics in identifying novel N-responsive proteins and may provide potential markers for rice response to low N.

Environmental proteomics: applications of proteome profiling in environmental microbiology and biotechnology

In this review, we present the use of proteomics to advance knowledge in the field of environmental biotechnology, including studies of bacterial physiology, metabolism and ecology. Bacteria are widely applied in environmental biotechnology for their ability to catalyze dehalogenation, methanogenesis, denitrification and sulfate reduction, among others. Their tolerance to radiation and toxic compounds is also of importance. Proteomics has an important role in helping uncover the pathways behind these cellular processes. Environmental samples are often highly complex, which makes proteome studies in this field especially challenging. Some of these challenges are the lack of genome sequences for the vast majority of environmental bacteria, difficulties in isolating bacteria and proteins from certain environments, and the presence of complex microbial communities. Despite these challenges, proteomics offers a unique dynamic view into cellular function. We present examples of environmental proteomics of model organisms, and then discuss metaproteomics (microbial community proteomics), which has the potential to provide insights into the function of a community without isolating organisms. Finally, the environmental proteomics literature is summarized as it pertains to the specific application areas of wastewater treatment, metabolic engineering, microbial ecology and environmental stress responses.

Global Screening of Human Cord Blood Proteomes for Biomarkers of Toxic Exposure and Effect

BackgroundExposures of pregnant women to natural and manmade chemicals can lead to negative health effects in the baby, ranging from low birth weight to developmental defects. In some cases, diseases were postulated to have their basis in toxic exposure in utero or in early childhood. Therefore, an understanding of fetal responses to environmental exposures is essential. To that end, cord blood is a readily accessible biofluid whose proteomic makeup remains mostly unexplored when compared with that of adults.ObjectivesOur goal was an initial global assessment of the fetal serum proteome and for the identification of protein biomarkers indicative of toxic in utero exposures related to maternal cigarette smoking.MethodsDrawing from a repository of 300 samples, we selected umbilical cord blood sera from 12 babies born to six smokers and six nonsmokers and analyzed both sample pools by tandem mass spectrometry in conjunction with isobaric tags (iTRAQ) for protein quantification.ResultsWe identified 203 proteins, 17 of which were differentially expressed between the cigarette smoke–exposed and control populations. Most of the identified candidate biomarkers were biologically plausible, thereby underscoring the feasibility of screening neonates with global proteomic techniques for biomarkers of exposure and early biological effects triggered by in utero chemical exposures.ConclusionsThis validation study provides an initial view of the proteome of human cord blood sera; it demonstrates the feasibility of identifying therein by use of proteomics, biomarkers of environmental, toxic exposures.

Influence of clotting time on the protein composition of serum samples based on LC–MS data

Many large, disease-related biobanks of serum samples have been established prior to the widespread use of proteomics in biomarker research. These biobanks may contain relevant information about the disease process, response to therapy or patient classifications especially with respect to long-term follow-up that is otherwise very difficult to obtain based on newly initiated studies, particularly in the case of slowly developing diseases. An important parameter that may influence the composition of serum but that is often not exactly known is clotting time. We therefore investigated the influence of clotting time on the protein and peptide composition of serum by label-free and stable-isotope labeling techniques. The label-free analysis of trypsin-digested serum showed that the overall pattern of LC–MS data is not affected by clotting times varying from 2 to 8 h. However, univariate and multivariate statistical analyses revealed that proteins that are directly involved in blood clot formation, such as the clotting-derived fibrinopeptides, change significantly. This is most easily detected in the supernatant of acid-precipitated, immunodepleted serum. Stable-isotope labeling techniques show that truncated or phosphorylated forms of fibrinopeptides A and B increase or decrease depending on clotting time. These patterns can be easily recognized and should be taken into consideration when analyzing LC–MS data using serum sample collections of which the clotting time is not known. Next to the fibrinopeptides, leucine-rich alpha-2-glycoprotein (P02750) was shown to be consistently decreased in samples with clotting times of more than 1 h. For prospective studies, we recommend to let blood clot for at least 2 h at room temperature using glass tubes with a separation gel and micronized silica to accelerate blood clotting.

Novel substrates and functions for the ubiquitin-like molecule NEDD8

Genetic experiments have established an important role for the ubiquitin-like molecule NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) in the regulation of cell growth, viability and development. It is therefore essential to identify the molecular targets for the pathway. Until recently, the cullin family of proteins was characterized as the only substrates for NEDDylation. However, through either direct biological approaches or the use of proteomics, it is now evident that the NEDD8 proteome is more diverse than thought previously. The present review describes the biological significance of NEDDylation for the novel identified substrates and the emerging evidence for the co-operation between the ubiquitin and NEDD8 pathways to control protein function.

Push–pull theory: Using mechanotransduction to achieve tissue perfusion and wound healing in complex cases

Wound healing has evolved from gauze therapy to the use of proteomics, gene therapy, and cellular-based therapies in the short time span of 45 years. Education for health care providers has not kept pace with the logarithmic acceleration in technology development and treatment options. A patient with a non-healing wound requires a comprehensive work-up, including a focus on six primary points of interest. These points include the status of tissue perfusion, role of bacterial contamination, pressure applied to the tissue, the immune status of the host, co-morbid medical conditions including the patient's psychosocial status, and lastly, the status of the wound itself. Even after re-establishing macrovascular flow, many wounds either fail to improve or paradoxically worsen. Potential mechanisms for these unexpected findings include reperfusion injury, no-reflow, and the presence of stunned/hibernating tissue. Using the concept of mechanotransduction, the clinician can simulate normal pulsatile blood flow and re-establish adequate microvascular perfusion. Treatment regimens may include negative pressure therapy, electrical stimulation, ultrasound therapy, and other energy-based modalities.