Current Articles in the field of proteomics published online in scientific journals.
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On this page considered biochemistry journals:
Molecular & Cellular Proteomics - published by
Society for Biochemistry and Molecular Biology -
... will publish three types of original articles: research papers, database articles and technology development articles.
Journal of Proteome Research - published by
The American Chemical Society -
... integrates the fields of chemistry, mathematics, applied physics, biology, and medicine in order to better understand the function of proteins in biological systems.
Proteomics - published by
Wiley-Interscience -
... is the premier international source for information on all aspects of applications and technologies in proteomics. It provides the most comprehensive coverage in the field, spanning significant technical developments in all major gel and non-gel based proteomic platforms and their application in every area of life sciences.
Current research articles of the mentioned
journals:
Helen Kim, Mark B. Cope, Richie Herring, Gloria Robinson, Landon Wilson, Grier P. Page, and Stephen Barnes Web Release Date: Thu, 4 Sep 2008 00:00:00 EDT (Article) DOI: 10.1021/pr800121b
The extraordinarily stable, non-covalent interaction between avidin and biotin is one of the most commonly exploited tools in chemistry and biology. Methods for derivatization with biotin of a variety of molecules (in particular, proteins) have been introduced, in order to allow their efficient recovery, immobilization and detection with avidin-based reagents. The field has evolved very rapidly and the applications have become more and more sophisticated. Cell surface protein studies have enormously benefited from refinements of this technology. It is now possible to specifically biotinylate one single membrane protein or to fish out a membrane receptor bound to its ligand. The release of biotinylated molecules from the avidin-based reagents, however, may still represent a major problem, due to the stability of the complex. This review will examine the biotin-avidin technology for the study of cell surface proteins, discussing reagents and techniques as well as examples of applications in quantitative proteomics.
The quality and ease of proteomics analysis depends on the performance of the analytical tools used, and thus of the performances of the protein separation tools used to deconvolute complex protein samples. Among protein samples, membrane proteins are one of the most difficult sample classes, because of their hydrophobicity and embedment in the lipid bilayers. This review deals with the recent progresses and advances made in the separation of membrane proteins by 2-DE separating only denatured proteins. Traditional 2-D methods, i.e., methods using IEF in the first dimension are compared to methods using only zone electrophoresis in both dimensions, i.e., electrophoresis in the presence of cationic or anionic detergents. The overall performances and fields of application of both types of method is critically examined, as are future prospects for this field
For the proteomic study of mitochondrial membranes, documented high quality mitochondrial preparations are a necessity to ensure proper localization. Despite the state-of-the-art technologies currently in use, there is no single technique that can be used for all studies of mitochondrial membrane proteins. Herein, we use examples to highlight solubilization techniques, different chromatographic methods, and developments in gel electrophoresis for proteomic analysis of mitochondrial membrane proteins. Blue-native gel electrophoresis has been successful not only for dissection of the inner membrane oxidative phosphorylation system, but also for the components of the outer membrane such as those involved in protein import. Identification of PTMs such as phosphorylation, acetylation, and nitration of mitochondrial membrane proteins has been greatly improved by the use of affinity techniques. However, understanding of the biological effect of these modifications is an area for further exploration. The rapid development of proteomic methods for both identification and quantitation, especially for modifications, will greatly impact the understanding of the mitochondrial membrane proteome.
Embryonic stem cells (ESCs) can give rise to any adult cell type and thus offer enormous potential for regenerative medicine and drug discovery. Molecular biomarkers serve as valuable tools to classify and isolate ESCs and to monitor their differentiation state by antibody-based techniques. A number of biomarkers, such as certain cell surface antigens, are used to assign pluripotent ESCs; however, accumulating evidence suggests that ESCs are heterogeneous in morphology, phenotype and function, and are thereby classified into subpopulations characterized by multiple sets of molecular biomarkers. Biomarker discovery is also important for ESC biology to elucidate the molecular mechanisms that regulate pluripotency and differentiation. This review summarizes studies of ESC biomarker discovery. "Genome-wide" expression profiling of ESC mRNAs and proteins and direct analyses of the cell surface subproteome have demonstrated that ESCs express a diverse range of biomarkers, cell surface antigens, and signaling molecules found in different cell lineages, as well as a number of key molecules that assure "stemness". Clearly, future quantitative proteomics approaches will enhance our knowledge of the stage- and lineage-specific expression of the proteome and its temporal changes upon differentiation, and provide a more detailed view of nascent and clonally amplified ESCs.
Production of membrane proteins (MPs) is a challenging task as their hydrophobic nature and their specific requirements in cellular expression systems frequently prevent an efficient synthesis. Cell-free (CF) expression systems have been developed in recent times as promising tools by offering completely new approaches to synthesize MPs directly into artificial hydrophobic environments. A considerable variety of CF produced MPs has been characterized by functional and structural approaches and the high success rates and the rapidly accumulating data on quality and expression efficiencies increasingly attract attention. In addition, CF expression is a highly dynamic and versatile technique and new modifications for improved performance as well as for extended applications for the labeling, throughput expression and proteomic analysis of MPs are rapidly emerging.
The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.
Receptors represent an abundant class of integral membrane proteins that transmit information on various types of signals within the cell. Assemblages of receptors and their interacting proteins (receptor complexes) have emerged as important units of signal transduction for various types of receptors including G protein coupled, ligand-gated ion channel, and receptor tyrosine kinase. This review aims to summarize the major approaches and findings of receptor proteomics. Isolation and characterization of receptor complexes from cells has become common using the methods of immunoaffinity-, ligand-, and tag-based chromatography followed by MS for the analysis of enriched receptor preparations. In addition, tools such as stable isotope labeling have contributed to understanding quantitative properties and PTMs to receptors and their interacting proteins. As data from studies on receptor-protein interactions considerably expands, complementary approaches such as bioinformatics and computational biology will undoubtedly play a significant role in defining cellular and network functions for various types of receptor complexes. Findings from receptor proteomics may also shed light on the mechanism of action for pharmacological drugs and can be of value in understanding molecular pathologies of disease states.
Biological membranes form an essential barrier between living cells and their external environments, as well as serve to compartmentalize intracellular organelles within eukaryotes. The latter includes membranes that envelope the nucleus, the outer and inner membranes of the mitochondria, membrane cisternae complex of the ER, Golgi apparatus, as well as lysosomes and secretory vesicles. Depending on their localizations in the whole organism and also within the cell, these membranes have different, highly specialized functions. Although 30% of naturally occurring proteins are predicted to be embedded in biological membranes, membrane proteomics is traditionally understudied due to difficulties in solubilizing, separating, and identifying membrane proteins. Given the importance of membrane proteins in the various cellular processes listed in this review, as well as the roles they play in diseases and their potential as drug targets, it is imperative that this class of proteins be better studied. With the recent advancement in technology, it is expected that some of the difficulties in membrane proteomics will be overcome, yielding new data on membrane proteins.
Plant cells contain many membrane systems that are specially adapted to perform particular functions. In plant cells, the processing of signals that are involved in responses to biotic and abiotic stressors occurs in the plasma membrane. Therefore, characterization of the plasma membrane proteome can provide new insights into the functions of various plant membrane systems. Plant plasma membrane proteomics can also provide valuable information for plant-specific biological investigations. Despite recent advances in preparative and analytical techniques for plant plasma membrane proteins, the characterization of these proteins, particularly the hydrophobic ones, remains challenging. In this review, plant plasma membrane proteomics data compiled from the literature on Arabidopsis thaliana are presented. Initial attempts to determine the physiological significance of some proteins identified from plasma membrane proteomics in rice and other plants are also described from the results of our research.
The analysis of integral membrane proteins (IMPs) with mass spectrometry-centered technologies has undergone great progress during the past few years, allowing for the analysis of several hundreds of IMPs. In this study, we investigated three promising shotgun approaches for the identification of IMPs of the model organism Bacillus subtilis. One comprises a classical membrane preparation procedure with carbonate and high-ionic-strength buffers, followed by SDS-PAGE and LC-MS/MS analysis. The two others are based on enzymatic trimming of the crude membrane fraction either with trypsin or proteinase K and subsequent gel-free analysis. As a result, we observed the highest degree of complementarity between the gel-based and the proteinase K approach, since the first exclusively addresses soluble loops and domains of IMPs and gave rise to 8709 unique peptides, whereas the latter contributed 1180 unique peptide identifications from otherwise inaccessible transmembrane helices (TMHs). All three methods contribute significant numbers (381, 284, and 276, respectively) to the total of 527 IMP identifications from the membrane fraction of exponentially growing B. subtilis cells, thus representing approximately 69% of all transcribed IMPs.
1-D native electrophoresis is used for the separation of individual proteins, protein complexes, and supercomplexes. Stable and labile protein-protein interactions can be identified depending on detergent and buffer conditions. 1-D native gels are immediately applicable for in-gel detection of fluorescent-labeled proteins and for in-gel catalytic activity assays. 1-D native gels and blots are used to determine native mass and oligomeric state of membrane proteins. Protein extracts from 1-D native gels are used for generation of antibodies, for proteomic work, and for advanced structural investigations. 2-D separation of subunits of protein complexes by SDS-PAGE is mostly used for immunological and proteomic studies. Following the discussion of these general features, specific applications of native electrophoresis techniques in various research fields are highlighted: immunological and receptor studies, biogenesis and assembly of membrane protein complexes, protein import into organelles, dynamics of proteasomes, proteome and subproteome investigations, the identification and quantification of mitochondrial alterations in apoptosis, carcinogenesis, and neurodegenerative disorders like Parkinson's disease, Alzheimer's disease, and the vast variety of mitochondrial encephalomyopathies.
Ewoud J. J. van Velzen, Johan A. Westerhuis, John P. M. van Duynhoven, Ferdi A. van Dorsten, Huub C. J. Hoefsloot, Doris M. Jacobs, Suzanne Smit, Richard Draijer, Christine I. Kroner, and Age K. Smilde Web Release Date: Thu, 28 Aug 2008 00:00:00 EDT (Article) DOI: 10.1021/pr800145j
Ambrosius P. L. Snijders, Sayampong Pongdam, Stan J. Lambert, Christopher M. Wood, John P. Baldwin, and Mark J. Dickman Web Release Date: Thu, 28 Aug 2008 00:00:00 EDT (Article) DOI: 10.1021/pr800260a
Yanfei Wang, Xiaoping Ao, Huy Vuong, Meghana Konanur, Fred R. Miller, Steve Goodison, and David M. Lubman Web Release Date: Wed, 27 Aug 2008 00:00:00 EDT (Article) DOI: 10.1021/pr8002547
Danielle Caron, Éric Winstall, Yutaka Inaguma, Sébastien Michaud, Francine Lettre, Sylvie Bourassa, Isabelle Kelly, Guy G. Poirier, Robert L. Faure, and Robert M. Tanguay Web Release Date: Wed, 27 Aug 2008 00:00:00 EDT (Article) DOI: 10.1021/pr8002497
Samïrah Perally, E. James LaCourse, Alison M. Campbell, and Peter M. Brophy Web Release Date: Sat, 23 Aug 2008 00:00:00 EDT (Article) DOI: 10.1021/pr800395x
Shujia Dai, Yifeng Jia, Shiaw-Lin Wu, Jeff S. Isenberg, Lisa A. Ridnour, Russell W. Bandle, David A. Wink, David D. Roberts, and Barry L. Karger Web Release Date: Sat, 23 Aug 2008 00:00:00 EDT (Article) DOI: 10.1021/pr800376w
Raghothama Chaerkady, H C Harsha, Anuradha Nalli, Marjan Gucek, Perumal Vivekanandan, Javed Akhtar, Robert N Cole, Jessica Simmers, Richard D. Schulick, Sujay Singh, Michael Torbenson, Akhilesh Pandey, and Paul J. Thuluvath Web Release Date: Thu, 21 Aug 2008 00:00:00 EDT (Article) DOI: 10.1021/pr800197z
Heart diseases resulting in heart failure are among the leading causes of morbidity and mortality in the Western world and can result from either systemic disease (e.g., hypertensive heart disease, ischemic heart disease) or specific heart muscle disease (e.g., dilated cardiomyopathy/DCM). Subproteome analysis of such disease subsets affords a reduction in sample complexity, potentially revealing biomarkers of cardiac failure that would otherwise remain undiscovered in proteome wide studies. Label-free nanoscale LC-MS has been applied in this study to validate a Triton X-114-based phase enrichment method for cardiac membrane proteins. Annotation of the subcellular location combined with GRAVY score analysis indicates a clear separation between soluble and membrane-bound proteins with an enrichment of over 62% for this protein subset. LC-MS allowed confident identification and annotation of hydrophobic proteins in this control sample pilot study and demonstrates the power of the proposed technique to extract integral membrane-bound proteins. This approach should be applicable to a wider scale study of disease-associated changes in the cardiac membrane subproteome.
Imaging mass spectrometry (IMS) technology utilizes MALDI MS to map molecules of interest in thin tissue sections. In this study, we have evaluated the potential of MALDI IMS to study peptide expression patterns in the mouse pancreas under normal and pathological conditions, and to in situ identify peptides of interest using MS/MS. Different regions of the pancreas of both control and ob/ob mice were imaged, resulting in peptide-specific profiles. The distribution of ions of m/z 3120 and 3439 displayed a striking resemblance with Langerhans islet's histology and, following MS/MS fragmentation and database searching were identified as C-peptide of insulin and glicentin-related polypeptide, respectively. In addition, a significant increase of the 3120 peak intensity in the obese mice was observed. This study underscores the potential of MALDI IMS to study the contribution of peptides to pancreas pathology.
Considering the importance of proteins in the structure and function of the cell wall of Candida albicans, we analyzed the cell wall subproteome of this important human pathogen by LC coupled to MS (LC-MS) using different protein extraction procedures. The analyzed samples included material extracted by hydrogen fluoride-pyridine (HF-pyridine), and whole SDS-extracted cell walls. The use of this latter innovative procedure gave similar data as compared to the analysis of HF-pyridine extracted proteins. A total of 21 cell wall proteins predicted to contain a signal peptide were identified, together with a high content of potentially glycosylated Ser/Thr residues, and the presence of a GPI motif in 19 of them. We also identified 66 "atypical" cell wall proteins that lack the above-mentioned characteristics. After tryptic removal of the most accessible proteins in the cell wall, several of the same expected GPI proteins and the most commonly found "atypical" wall proteins were identified. This result suggests that proteins are located not only at the cell wall surface, but are embedded within the cell wall itself. These results, which include new identified cell wall proteins, and comparison of proteins in blastospore and mycelial walls, will help to elucidate the C. albicans cell wall architecture.
The characterization and localization of peptides and proteins in tissues provides information that aids in understanding their function and in characterizing disease states. Over the past decades, the use of MS for the profiling and imaging of biological compounds from tissues has evolved into a powerful modality to accomplish these studies. One recently described sampling approach, the stretched sample method (Monroe, E. B. et al.., Anal. Chem. 2006, 78, 6826-6832), places a tissue section onto an array of glass beads embedded on a Parafilm M membrane. When the membrane is stretched, it separates the tissue section into thousands of cell-sized pieces for tissue profiling by MALDI-MS. The physical separation between beads eliminates analyte redistribution during matrix application and allows long analyte extraction periods without loss of spatial resolution. Here, we enhance this sampling approach by introducing algorithms that enable the reconstruction of ion images from these stretched samples. As the first step, a sample-tailored data acquisition method is devised to obtain mass spectra exclusively from the beads, thereby reducing the time, instrument resources, and data handling required for such MS imaging (MSI) experiments. Next, an image reconstruction algorithm matches data acquired from the stretched sample to the initial bead locations. The efficacy of this method is demonstrated using peptide-coated beads with known peptide distributions and appears well-suited to the MSI of heterogeneous tissue samples.
Laser ablation (LA) ICP-MS has been developed as a new tool for imaging of cancer biomarkers in tissue sections. The distribution of two breast cancer-associated proteins, MUC-1 and HER2 was studied based on multiple line rastering of tissue sections and measurement of relevant Au/Ag tagged antibodies bound to the tissue. Comparisons with optical microscopy indicated extremely high sensitivity for the LA technique and sufficiently good resolution to permit fine scale feature mapping at the cellular level. Application to the quantitative assessment of HER2 expression in tissue microarrays was demonstrated.
Imaging using MS has the potential to deliver highly parallel, multiplexed data on the specific localization of molecular ions in tissue samples directly, and to measure and map the variations of these ions during development and disease progression or treatment. There is an intrinsic potential to be able to identify the biomarkers in the same experiment, or by relatively simple extension of the technique. Unlike many other imaging techniques, no a priori knowledge of the markers being sought is necessary. This review concentrates on the use of MALDI-MS for MS imaging (MSI) of proteins and peptides, with an emphasis on mammalian tissue. We discuss the methodologies used, their potential limitations, overall experimental considerations and progress that has been made towards establishing MALDI-MSI as a routine technique for the spatially resolved measurement of peptides and proteins. As well as determining the local abundance of individual molecular ions, there is the potential to determine their identity within the same experiment using relatively simple extensions of the basic techniques. In this way MSI offers an important opportunity for biomarker discovery and identification.
This study applied yolk immunoglobulins immunoaffinity separation and MALDI-TOF MS for clinical proteomics of congenital disorders of glycosylation (CDG) and secondary glycosylation disorders [galactosemia and hereditary fructose intolerance (HFI)]. Serum transferrin (Tf) and [alpha]1-antitrypsin (AAT) that are markers for CDG, were purified sequentially to obtain high-quality MALDI mass spectra to differentiate single glycoforms of the native intact glycoproteins. The procedure was found feasible for the investigation of protein macroheterogeneity due to glycosylation site underoccupancy then ensuing the characterization of patients with CDG group I (N-glycan assembly disorders). Following PNGase F digestion of the purified glycoprotein, the characterization of protein microheterogeneity by N-glycan MS analysis was performed in a patient with CDG group II (processing disorders). CDG-Ia patients showed a typical profile of underglycosylation where the fully glycosylated glycoforms are always the most abundant present in plasma with lesser amounts of partially and unglycosylated glycoforms in this order. Galactosemia and HFI are potentially fatal diseases, which benefit from early diagnosis and prompt therapeutic intervention. In symptomatic patients with galactosemia and in those with HFI, MALDI MS of Tf and AAT depicts a hypoglycosylation profile with a significant increase of underglycosylated glycoforms that reverses by dietary treatment, representing a clue for diagnosis and treatment monitoring.
A novel method for high-throughput proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMA) is described using on-tissue tryptic digestion followed by MALDI imaging MS. A TMA section containing 112 needle core biopsies from lung-tumor patients was analyzed using MS and the data were correlated to a serial hematoxylin and eosin (H&E)-stained section having various histological regions marked, including cancer, non-cancer, and normal ones. By correlating each mass spectrum to a defined histological region, statistical classification models were generated that can sufficiently distinguish biopsies from adenocarcinoma from squamous cell carcinoma biopsies. These classification models were built using a training set of biopsies in the TMA and were then validated on the remaining biopsies. Peptide markers of interest were identified directly from the TMA section using MALDI MS/MS sequence analysis. The ability to detect and characterize tumor marker proteins for a large cohort of FFPE samples in a high-throughput approach will be of significant benefit not only to investigators studying tumor biology, but also to clinicians for diagnostic and prognostic purposes.
The advent of metal cluster as a primary ion source in the late 1980s, made it feasible to probe surfaces for complex organic structures due to a reduced in-source fragmentation, and opened the door to the direct analysis of biological samples. Despite the mass range measurable by TOF-secondary ion MS (SIMS) still being rather limited, the information obtained from cells and tissues comes together with the technical innovations introduced in the last decade. In this article, we give a brief overview of the technique itself and make some emphasis on the advances in the last three years in the analysis of biological surfaces, particularly those with direct implication in the biomedical field; reviewing what kind of information this instrumentation will add to current tool in pathology.
The application of MS to imaging, or MS imaging (MSI), allows for the direct investigation of tissue sections to identify biological compounds and determine their spatial distribution. We present an approach to MSI that combines secondary ion MS (SIMS) and MALDI MS for the imaging and analysis of rat spinal cord sections, thereby enhancing the chemical coverage obtained from an MSI experiment. The spinal cord is organized into discrete, anatomically defined areas that include motor and sensory networks composed of chemically diverse cells. The MSI data presented here reveal the spatial distribution of multiple phospholipids, proteins, and neuropeptides obtained within single, 20 [mu]m sections of rat spinal cord. Analyte identities are initially determined by primary mass match and confirmed in follow-up experiments using LC MS/MS from extracts of adjacent spinal cord sections. Additionally, a regional analysis of differentially localized signals serves to rapidly screen compounds of varying intensities across multiple spinal regions. These MSI analyses reveal new insights into the chemical architecture of the spinal cord and set the stage for future imaging studies of the chemical changes induced by pain, anesthesia, and drug tolerance.
We have used MALDI-MS imaging (MALDI-MSI) to monitor the time dependent appearance and loss of signals when tissue slices are brought rapidly to room temperature for short to medium periods of time. Sections from mouse brain were cut in a cryostat microtome, placed on a MALDI target and allowed to warm to room temperature for 30 s to 3 h. Sections were then refrozen, fixed by ethanol treatment and analysed by MALDI-MSI. The intensity of a range of markers were seen to vary across the time course, both increasing and decreasing, with the intensity of some markers changing significantly within 30 s and markers also showed tissue location specific evolution. The markers resulting from this autolysis were compared directly to those that evolved in a comparable 16 h on-tissue trypsin digest, and the markers that evolved in the two studies were seen to be substantially different. These changes offer an important additional level of location-dependent information for mapping changes and seeking disease-dependent biomarkers in the tissue. They also indicate that considerable care is required to allow comparison of biomarkers between MALDI-MSI experiments and also has implications for the standard practice of thaw-mounting multiple tissue sections onto MALDI-MS targets.
Eszter Bohus, Muireann Coen, Hector C. Keun, Timothy M. D. Ebbels, Olaf Beckonert, John C. Lindon, Elaine Holmes, Béla Noszál, and Jeremy K. Nicholson Web Release Date: Tue, 19 Aug 2008 00:00:00 EDT (Article) DOI: 10.1021/pr800407j
Patricia Alfonso, Marta Cañamero, Francisco Fernández-Carbonié, Antonio Núñez, and J. Ignacio Casal Web Release Date: Sat, 16 Aug 2008 00:00:00 EDT (Article) DOI: 10.1021/pr800152u
Adam Burgener, Julie Boutilier, Charles Wachihi, Joshua Kimani, Michael Carpenter, Garrett Westmacott, Keding Cheng, Terry B. Ball, and Francis Plummer Web Release Date: Sat, 16 Aug 2008 00:00:00 EDT (Article) DOI: 10.1021/pr800406r
Robbie Montgomery, Hanna Shay, Matthew McCarroll, and Luke Tolley Web Release Date: Sat, 16 Aug 2008 00:00:00 EDT (Technical Note) DOI: 10.1021/pr800354f
Surfactins are a family of heptacyclopeptides in which the C-terminal carbonyl is linked with the [beta]-hydroxy group of a fatty acid acylating the N-terminal function of a glutamic acid residue. The fatty acyl chain is 12-16 carbon atoms long. These compounds, which are secreted by the Gram-positive bacterium Bacillus subtilis in stationary phase in liquid cultures, play an important role in swarming communities on the surface of agar media in the formation of dendritic patterns. TOF secondary ion MS (TOF-SIMS) imaging was used to map surfactins within 16-17 h swarming patterns, with a 2 [mu]m spatial resolution. Surfactins were mainly located in the central mother colony (the site of initial inoculation), in a 'ring' surrounding the pattern and along the edges of the dendrites. In the mother colony and the interior of the dendrites, surfactins with shorter chain lengths are present, whereas in the ring surrounding the swarm community and between dendrites, surfactins with longer fatty acyl chain lengths were found. A quantitative analysis by MALDI-TOF MS showed a concentration gradient of surfactin from the mother colony to the periphery. The concentration of surfactin was [sim]400 pmol/mL in the mother colony and [sim]10 pmol/mL at the base of the dendrites, decreasing to 2 pmol/mL at their tips.
Archival formalin-fixed paraffin-embedded (FFPE) tissues are a powerful tool for examining the clinical course of diseases. These specimens represent an incredible mine of valuable clinical and biological information for proteomic investigation. MALDI-TOF imaging MS (MALDI-IMS) is a protein profiling technique which enables the direct sampling of histological section; however, the quality of molecular data are strongly influenced by the tissue preparation condition. In fact, in previous years most of the studies employing such a technological platform have been conducted using cryo-preserved tissues. We have developed an in vitro approach using "tissue surrogate" samples in order to explore different protein unlocking procedures which might enable a suitable recovery of polypeptides for MS analysis. The developed protocols have been compared both by MALDI-TOF MS and nLC-MSE analysis either on surrogate samples or on FFPE specimen from human breast cancer. The collected evidence has been applied for the preparation of FFPE tissue sections following MALDI-IMS analysis. Our results outline the possibility to obtain valuable peptide mass spectra profiles form FFPE preparations by applying a combined two steps procedure of heat induced antigen retrieval (HIAR) in presence of EDTA and on target trypsin hydrolysis. A multivariate statistical evaluation is presented and discussed according to molecular spatial distributions and tissue morphology.
This paper describes the development of laser desorption 7.87 eV vacuum UV (VUV) postionization MS to detect antibiotics within intact bacterial colony biofilms. As >99% of the molecules ejected by laser desorption are neutrals, VUV photoionization of these neutrals can provide significantly increased signal as compared to the detection of directly emitted ions. Postionization with VUV radiation from the molecular fluorine laser single photon ionizes laser desorbed neutrals with ionization potentials below the 7.87 eV photon energy. Antibiotics with structures indicative of sub-7.87 eV ionization potentials were examined for their ability to be detected by 7.87 eV laser desorption postionization MS. Tetracycline, sulfadiazine, and novobiocin were successfully detected neat as dried films physisorbed on porous silicon oxide substrates. Tetracycline and sulfadiazine were then detected within intact Staphylococcus epidermidis colony biofilms, the former with LOD in the micromolar concentration range.
The field of organellar proteomics has emerged as an attempt to minimize the complexity of the proteomics data obtained from whole cell and tissue extracts while maximizing the resolution on the protein composition of a single subcellular compartment. Standard methods involve lengthy density-based gradient and/or immunoaffinity purification steps followed by extraction, 1-DE or 2-DE, gel staining, in-gel tryptic digestion, and protein identification by MS. In this paper, we present an alternate approach to purify subcellular organelles containing a fluorescent reporter molecule. The gel-free procedure involves fluorescence-assisted sorting of the secretory granules followed by gentle extraction in a buffer compatible with tryptic digestion and MS. Once the subcellular organelle labeled, this procedure can be done in a single day, requires no major modification to any instrumentation and can be readily adapted to the study of other organelles. When applied to corticotrope secretory granules, it led to a much enriched granular fraction from which numerous proteins could be identified through MS.
Heterosis describes the superior performance of heterozygous F1-hybrids compared to their homozygous parental inbred lines. Heterosis is already manifested during early maize (Zea mays L.) primary root development. In this study, the most abundant soluble proteins have been investigated before the phenotypic manifestation of heterosis in 3.5-day-old primary roots in the flint inbred line UH002, the dent inbred line UH301 and the corresponding hybrid UH301×UH002. In CBB-stained 2-DE gels, 150 of 304 detected proteins (49%) were accumulated in a nonadditive fashion in the hybrid compared to the average of their parental inbred lines (Student's t-test: p < 0.05). Remarkably, expression of 51% (76/150) of the nonadditively accumulated proteins exceeded the high parent or was below the low parent. ESI-MS/MS identified 75 of the 76 proteins that belonged to these expression classes. The most abundant functional classes among the 75 proteins that were encoded by 60 different genes were metabolism (58%) and disease and defense (19%). Nonadditive protein accumulation in primary roots of maize hybrids might be associated with heterosis manifestation. Identification of these proteins could therefore contribute to the better understanding of the molecular basis of heterosis.
A 2-D DIGE approach allowed the characterization of the intramacrophagic proteome of the intracellular pathogen Brucella suis at the late stage of in vitro infection by efficient discrimination between bacterial and host cell proteins. Using a subtraction model, a total of 168 proteins showing altered concentrations in comparison with extracellularly grown, stationary-phase bacteria were identified. The majority of the 44 proteins significantly regulated at this stage of infection were involved in bacterial metabolism and 40% were present in lowered concentrations, supporting the hypothesis of an adaptive response by quantitative reduction of processes participating in energy, protein, and nucleic acid metabolism. In the future, the 2-D DIGE-based approach will permit to decipher specifically and quantitatively the intracellular proteomes of various pathogens during adaptation to their specific host cell environments.
Benjamin A. Garcia, C. Eric Thomas, Neil L. Kelleher, and Craig A. Mizzen Web Release Date: Thu, 14 Aug 2008 00:00:00 EDT (Article) DOI: 10.1021/pr800044q
Protein synthesis is one of the most important reactions in the cell. Recent experimental studies indicated that this complex reaction can be achieved with a minimum complement of 36 proteins and ribosomes by reconstituting an Escherichia coli-based in vitro translation system with these protein components highly purified on an individual basis. From the protein-protein interaction (PPI) network of E. coli proteins, these minimal protein components are known to interact physically with large numbers of proteins. However, it is unclear what fraction of E. coli proteins are linked functionally with the minimal protein synthesis system. We investigated the effects of each of the 4194 E. coli ORF products on the minimal protein synthesis system; at least 12% of the entire ORF products, a significant fraction of the gene product of E. coli, affect the activity of this system. Furthermore 34% of these functional modifiers present in the PPI network were shown by mapping to be directly linked (i.e. to interact physically) with the minimal components of the PPI network. Topological analysis of the relationships between modifiers and the minimal components in the PPI network indicated clustering of the minimal components. The modifiers showed no such clustering, indicating that the location of functional modifiers is spread across the PPI network rather than clustering close to the minimal protein components. These observations may reflect the evolutionary process of the protein synthesis system.
Caspase-6 activation occurs early in Alzheimer disease and sometimes precedes the clinical manifestation of the disease in aged individuals. The active Caspase-6 is localized in neuritic plaques, in neuropil threads, and in neurofibrillary tangles containing neurons that are not morphologically apoptotic in nature. To investigate the potential consequences of the activation of Caspase-6 in neurons, we conducted a proteomics analysis of Caspase-6-mediated cleavage of human neuronal proteins. Proteins from the cytosolic and membrane subcellular compartments were treated with recombinant active Caspase-6 and compared with undigested proteins by two-dimensional gel electrophoresis. LC/MS/MS analyses of the proteins that were cleaved identified 24 different potential protein substrates. Of these, 40% were cytoskeleton or cytoskeleton-associated proteins. We focused on the cytoskeleton proteins because these are critical for neuronal structure and function. Caspase-6 cleavage of -Tubulin, -Actinin-4, Spinophilin, and Drebrin was confirmed. At least one Caspase-6 cleavage site was identified for Drebrin, Spinophilin, and -Tubulin. A neoepitope antiserum to -Tubulin cleaved by Caspase-6 immunostained neurons, neurofibrillary tangles, neuropil threads, and neuritic plaques in Alzheimer disease and co-localized with active Caspase-6. These results imply that the early and neuritic activation of Caspase-6 in Alzheimer disease could disrupt the cytoskeleton network of neurons, resulting in impaired neuronal structure and function in the absence of cell death. This study provides novel insights into the pathophysiology of Alzheimer disease.
Hydrogen peroxide (H2O2) plays a dual role in plants as the toxic by-product of normal cell metabolism and as a regulatory molecule in stress perception and signal transduction. However, a clear inventory as to how this dual function is regulated in plants is far from complete. In particular, how plants maintain survival under oxidative stress via adjustments of the intercellular metabolic network and antioxidative system is largely unknown. To investigate the responses of rice seedlings to H2O2 stress, changes in protein expression were analyzed using a comparative proteomics approach. Treatments with different concentrations of H2O2 for 6 h on 12-day-old rice seedlings resulted in several stressful phenotypes such as rolling leaves, decreased photosynthetic and photorespiratory rates, and elevated H2O2 accumulation. Analysis of ~2000 protein spots on each two-dimensional electrophoresis gel revealed 144 differentially expressed proteins. Of them, 65 protein spots were up-regulated, and 79 were down-regulated under at least one of the H2O2 treatment concentrations. Furthermore 129 differentially expressed protein spots were identified by mass spectrometry to match 89 diverse protein species. These identified proteins are involved in different cellular responses and metabolic processes with obvious functional tendencies toward cell defense, redox homeostasis, signal transduction, protein synthesis and degradation, photosynthesis and photorespiration, and carbohydrate/energy metabolism, indicating a good correlation between oxidative stress-responsive proteins and leaf physiological changes. The abundance changes of these proteins, together with their putative functions and participation in physiological reactions, produce an oxidative stress-responsive network at the protein level in H2O2-treated rice seedling leaves. Such a protein network allows us to further understand the possible management strategy of cellular activities occurring in the H2O2-treated rice seedling leaves and provides new insights into oxidative stress responses in plants.
G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are major drug targets. Recent progress has shown that GPCRs are part of large protein complexes that regulate their activity. We present here a generic approach for identification of these complexes that is based on the use of receptor subdomains and that overcomes the limitations of currently used genetics and proteomics approaches. Our approach consists of a carefully balanced combination of chemically synthesized His6-tagged baits, immobilized metal affinity chromatography, one- and two-dimensional gel electrophoresis separation and mass spectrometric identification. The carboxyl-terminal tails (C-tails) of the human MT1 and MT2 melatonin receptors, two class A GPCRs, were used as models to purify protein complexes from mouse brain lysates. We identified 32 proteins that interacted with the C-tail of MT1, 14 proteins that interacted with the C-tail of MT2, and eight proteins that interacted with both C-tails. Several randomly selected proteins were validated by Western blotting, and the functional relevance of our data was further confirmed by showing the interaction between the full-length MT1 and the regulator of G protein signaling Z1 in transfected HEK 293 cells and native tissue. Taken together, we have established an integrated and generic purification strategy for the identification of high quality and functionally relevant GPCR-associated protein complexes that significantly widens the repertoire of available techniques.
In systemic amyloidoses, widespread deposition of protein as amyloid causes severe organ dysfunction. It is necessary to discriminate among the different forms of amyloid to design an appropriate therapeutic strategy. We developed a proteomics methodology utilizing two-dimensional polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry and peptide mass fingerprinting to directly characterize amyloid deposits in abdominal subcutaneous fat obtained by fine needle aspiration from patients diagnosed as having amyloidoses typed as immunoglobulin light chain or transthyretin. Striking differences in the two-dimensional gel proteomes of adipose tissue were observed between controls and patients and between the two types of patients with distinct, additional spots present in the patient specimens that could be assigned as the amyloidogenic proteins in full-length and truncated forms. In patients heterozygotic for transthyretin mutations, wild-type peptides and peptides containing amyloidogenic transthyretin variants were isolated in roughly equal amounts from the same protein spots, indicative of incorporation of both species into the deposits. Furthermore novel spots unrelated to the amyloidogenic proteins appeared in patient samples; some of these were identified as isoforms of serum amyloid P and apolipoprotein E, proteins that have been described previously to be associated with amyloid deposits. Finally changes in the normal expression pattern of resident adipose proteins, such as down-regulation of B-crystallin, peroxiredoxin 6, and aldo-keto reductase I, were observed in apparent association with the presence of amyloid, although their levels did not strictly correlate with the grade of amyloid deposition. This proteomics approach not only provides a way to detect and unambiguously type the deposits in abdominal subcutaneous fat aspirates from patients with amyloidoses but it may also have the capability to generate new insights into the mechanism of the diseases by identifying novel proteins or protein post-translational modifications associated with amyloid infiltration.
In many studies, particularly in the field of systems biology, it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quantitative approach by which predetermined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quantitative Analysis by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amounts of plasma. The resulting quantitative protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.
Biomolecule phosphorylation by protein kinases is a fundamental cell signaling process in all living cells. Following the comprehensive cataloguing of the protein kinase complement of the human genome (Manning, G., Whyte, D. B., Martinez, R., Hunter, T., and Sudarsanam, S. (2002) The protein kinase complement of the human genome. Science 298, 1912–1934), this review will detail the state-of-the-art human and mouse kinase proteomes as provided in the UniProtKB/Swiss-Prot protein knowledgebase. The sequences of the 480 classical and up to 24 atypical protein kinases now believed to exist in the human genome and 484 classical and up to 24 atypical kinases within the mouse genome have been reviewed and, where necessary, revised. Extensive annotation has been added to each entry. In an era when a wealth of new databases is emerging on the Internet, UniProtKB/Swiss-Prot makes available to the scientific community the most up-to-date and in-depth annotation of these proteins with access to additional external resources linked from within each entry. Incorrect sequence annotations resulting from errors and artifacts have been eliminated. Each entry will be constantly reviewed and updated as new information becomes available with the orthologous enzymes in related species being annotated in a parallel effort and complete kinomes being completed as sequences become available. This ensures that the mammalian kinomes available from UniProtKB/Swiss-Prot are of a consistently high standard with each separate entry acting both as a valuable information resource and a central portal to a wealth of further detail via extensive cross-referencing.
Type 2 diabetes (T2D) arises when pancreatic β-cells fail to compensate for systemic insulin resistance with appropriate insulin secretion. However, the link between insulin resistance and β-cell failure in T2D is not fully understood. To explore this association, we studied transgenic MKR mice that initially develop insulin resistance in skeletal muscle but by 8 weeks of age have T2D. In the present study, global islet protein and gene expression changes were characterized in diabetic MKR versus non-diabetic control mice at 10 weeks of age. Using a quantitative proteomics approach (isobaric tags for relative and absolute quantification (iTRAQ)), 159 proteins were differentially expressed in MKR compared with control islets. Marked up-regulation of protein biosynthesis and endoplasmic reticulum stress pathways and
