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At least 433 records · Page 24Linked to original sources
PubMed2004

Lectin-based structural glycomics: glycoproteomics and glycan profiling.

Structural glycomics (SG) plays a fundamental part of concurrent glycobiology aiming at comprehensive elucidation of glycan functions ( i.e. , functional glycomics) in the context of post-genome sciences. The SG project started in April 2003 and will continue for 3 years in the framework of NEDO (New Energy and Industrial Technology Organization) under the METI (the Ministry of Economy, Trade, and Industry), Japan. The main purpose of the project is the development of high-throughput and robust machines, which should greatly contribute to the structural analysis of complex glycans. In this chapter, 2 major research items, i.e. , (1) glycoproteomics, which enables comprehensive analysis of glycoproteins, and (2) "glycan profiling" by means of lectins, are described. For the latter, frontal affinity chromatography has been adopted as a starting tool for comprehensive analysis of the interaction of 100 lectins and 100 oligosaccharides under the concept of "hect-by-hect," which refers to 100 x 100.

Animals↗
PubMed2004

Towards higher-throughput membrane protein production for structural genomics initiatives.

Integral membrane proteins present unparalleled challenges for structural genomics programs. Samples from this class of proteins are not only difficult to produce in quantities sufficient for analysis by X-ray diffraction or NMR, but their hydrophobic properties add extra dimension to their purification and subsequent crystallization. New systems that seek to tackle the production problems are in development. In our laboratory, one such strategy exploits the unique physiology of the Rhodobacter species of photosynthetic bacteria where we have designed an overexpression system that coordinates the heterologous production of targeted hydrophobic proteins with nascent, unfilled membranes that can be used to harbor them. In this study, we describe the means by which purification of recombinant membrane proteins produced in such a fashion can be purified efficiently from Rhodobacter membranes using relatively higher-throughput, semi-automated methods. These protocols utilize a state-of-the-art FPLC system for affinity chromatography, followed by gel filtration or ion exchange chromatography to enhance purity for crystallization attempts. The Rhodobacter expression system coupled with the semi-automation of purification steps represents an advance towards the development of a strategy for obtaining structures for membrane proteins at a more rapid pace.

Chromatography, Affinity↗
PubMed2004

Automation of protein purification for structural genomics.

A critical issue in structural genomics, and in structural biology in general, is the availability of high-quality samples. The additional challenge in structural genomics is the need to produce high numbers of proteins with low sequence similarities and poorly characterized or unknown properties. 'Structural-biology-grade' proteins must be generated in a quantity and quality suitable for structure determination experiments using X-ray crystallography or nuclear magnetic resonance (NMR). The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. The purification procedure must yield a homogeneous protein and must be highly reproducible in order to supply milligram quantities of protein and/or its derivative containing marker atom(s). At the Midwest Center for Structural Genomics we have developed protocols for high-throughput protein purification. These protocols have been implemented on AKTA EXPLORER 3D and AKTA FPLC 3D workstations capable of performing multidimensional chromatography. The automated chromatography has been successfully applied to many soluble proteins of microbial origin. Various MCSG purification strategies, their implementation, and their success rates are discussed in this paper.

Automation↗
PubMed2005

Concanavalin A-captured glycoproteins in healthy human urine.

Both the urinary proteome and its glycoproteome can reflect human health status, and more directly, functions of kidney and urinary tracts. Because the high abundance protein albumin is not N-glycosylated, the urine N-glycoprotein enrichment procedure could deplete it, and urine proteome could thus provide a more detailed protein profile in addition to glycosylation information especially when albuminuria occurs in some kidney diseases. In terms of describing the details of urinary proteins, the urine glycoproteome is even a better choice than the proteome itself. Pooled urine samples from healthy volunteers were collected and acetone-precipitated for proteins. N-Linked glycoproteins enriched with concanavalin A affinity purification were separated and analyzed by SDS-PAGE-reverse phase LC/MS/MS or two-dimensional LC/MS/MS. A total of 225 urinary proteins were identified based on two-hit criteria with reliability over 97% for each peptide. Among these proteins, 94 were identified in previous urine proteome works, 150 were annotated as glycoproteins in Swiss-Prot, and 43 were predicted as glycoproteins by NetNGlyc 1.0. A number of known biomarkers and disease-related glycoproteins were identified. Because changes in protein quantity or the glycosylation status can lead to changes in the concanavalin A-captured glycoprotein profile, specific urine glycoproteome patterns might be observed for specific pathological conditions as multiplex urinary biomarkers. Knowledge of the urine glycoproteome is important in understanding kidney and body function.

Concanavalin A↗
PubMed2002

Analytical aspects of mass spectrometry and proteomics.

Mass spectrometry plays an essential role in proteomics analysis and research. In recent years, it has been increasingly recognized that a key to proteomics using mass spectrometry relies not only on the instrument itself, but also on the analytical strategies and front-end sample-handling techniques. The advances of separations and mass spectrometry are having an increasing impact on the discovery of disease biomarkers and the understanding of cellular processes.

Affinity Labels↗
PubMed2026

Dynamic lysine acetylation and succinylation of platelet proteins regulates platelet storage lesion: mechanistic insights from multi-omics.

OBJECTIVES: Platelet storage lesion (PSL) severely impairs platelet function during storage, presenting a major hurdle in transfusion medicine; however, the dynamic interplay between global proteomic changes and post-translational modifications (PTMs) underlying these functional deteriorations remains insufficiently characterized. Here, we report the first comprehensive multi-omics analysis integrating global proteomics, acetylomics, and succinylomics to dissect the molecular dynamics during platelet storage. METHODS: We performed quantification of global proteomics, acetylome and succinylome based on TMT-labeled LC-MS/MS analysis, combined with antibody-affinity enrichment and purification. Dynamic molecular changes and functional transformation of platelet were also characterized under proper conditions stored for 1, 3, 5, 7 days, respectively. RESULTS: We systematically characterized 3,609 proteins, 1,308 acetylation sites, and 1,947 succinylation sites across multiple storage time points (D1, D3, D5, D7). We distinct temporal patterns of post-translational modifications, with succinylation showing more extensive coverage than acetylation in platelets. Pathway enrichment analysis revealed extensive metabolic reprogramming involving complement activation, energy metabolism, and cellular detoxification processes. The identification of specific motif patterns provided mechanistic insights into the functional specificity of these modifications. Random forest machine learning identified 20 core regulatory proteins representing critical nodes in PSL development. Furthermore, we employed real - time quantitative polymerase chain reaction (RT - QPCR) to measure the expression levels of key genes related to platelet function and PTM - associated pathways. CONCLUSION: By mapping the interplay between proteomic abundance shifts and PTM dynamics, this study provides a multidimensional understanding of PSL, establishing a foundational framework for optimizing storage protocols and enhancing transfusion safety.

Blood Platelets↗
PubMed2005

Single-step affinity purification of toxic and non-toxic proteins on a fluidics platform.

Single-step fusion-based affinity purification of proteins with pH-controllable linkers was carried out in a fluidic device. The linkers were previously derived from self-splicing protein elements called inteins. Two different linkers were generated to solve two distinct separation problems: one for rapid single-step affinity purification of a wide range of proteins, and the other specifically for the purification of cytotoxic proteins. Scale-down factors of 185 resulted in separations in a 27 microl bed-volume. A rotating CD format was chosen because of its simplicity in effecting fluid movement through centrifugal force without the complications associated with electro-osmosis and other pumping methods. The design and fabrication of the fluidic device and the protein purification process are described. This work, which demonstrates the purification of active proteins by two distinct fluidic separations, is widely applicable to small-scale massively parallel proteomic separations.

Chromatography, Affinity↗
PubMed2002

Affinity capturing and gene assignment of soluble glycoproteins produced by the nematode Caenorhabditis elegans.

Protein glycosylation is a central issue for post-genomic (proteomic) sciences. We have taken a systematic approach for analyzing soluble glycoproteins produced in the nematode Caenorhabditis elegans. The approach aims at assigning (i) genes that encode glycoproteins, (ii) sites where glycosylation occurs, and (iii) types of attached glycan structures. A soluble extract of C. elegans, as a starting material, was applied first to a concanavalin A (ConA) column (specific for high-mannose type N-glycans), and then the flow-through fraction was applied to a galectin LEC-6 (GaL6) column (specific for complex-type N-glycans). The adsorbed glycoproteins were digested with lysylendopeptidase, and the resultant glycopeptides were selectively recaptured with the same lectin columns. The glycopeptides were separated by reversed-phase chromatography and then subjected to sequence determination. As a result, 44 and 23 glycopeptides captured by the ConA and GaL6 columns, respectively, were successfully analyzed and assigned to 32 and 16 corresponding genes, respectively. For these glycopeptides, 49 N-glycosylation sites were experimentally confirmed, whereas 21 sites remained as potential sites. Of the identified genes, about 80% had apparent homologues in other species, as represented by typical secreted proteins. However, the two sets of genes assigned for the ConA and GaL6-recognized glycopeptides showed only 1 overlap with each other. Proof of the practical applicability of the glyco-catch method to a model organism, C. elegans, directs us to explore more complex multicellular organisms.

Adsorption↗
PubMed2006

Towards high-throughput characterization of small molecule mechanisms of action.

Drug discovery is hampered by the lack of general strategies to characterize the mechanisms of action and intracellular targets of bioactive small molecules. Genomics and proteomics promise to aid in this process. Genome-wide approaches in yeast have proven useful to infer the targets and target pathways of small molecules. These approaches are being systematically transferred into mammalian cell culture systems in order to interrogate more complex pathways in a more relevant setting. Advances in proteomics and in vivo genetic screening in multicellular model organism systems are also becoming increasingly powerful and amenable to high-throughput. Current methodologies and technologies are discussed, including how these global approaches complement affinity-based target identification strategies.

Animals↗
PubMed2026

Chemo-selective proteomics in microbial systems.

SUMMARYOver the past two decades, the field of bioorthogonal chemistry has transitioned from emerging to an established cornerstone of scientific inquiry. In parallel, advances in microbial and host-microbe research have highlighted the need for functional approaches that extend beyond genomic and transcriptomic analyses to directly interrogate protein-level activity. Despite this need, proteomic strategies capable of resolving dynamic, heterogeneous, and low-abundance protein populations remain underdeveloped in microbial systems. This review highlights the convergence of chemo-selective proteomic technologies with microbial biology, focusing on bioorthogonal non-canonical amino acid tagging (BONCAT), activity- or affinity-based protein profiling, and bioorthogonal post-translational modifications, and comments on possibilities for novel applications for the use of click chemistry-based tools in the functional interrogation of microbial systems. Together, these strategies enable spatiotemporal resolution of protein synthesis, selective profiling of microbial subpopulations, and direct characterization of protein activity and regulation in complex biological contexts, including single-species cultures, host-associated environments, and polymicrobial communities. Continued development and utilization of these technologies will enable deeper mechanistic insight into how microbial systems function and respond to environmental and host-derived cues.

bioorthogonal chemistry↗
PubMed2004

Protein profiling of cervical cancer by protein-biochips: proteomic scoring to discriminate cervical cancer from normal cervix.

Analysis of multiple proteins is thought to be essential for establishment of signature proteomic patterns that may distinguish cancer from non-cancer. Surface-enhanced laser desorption/ionization (SELDI) is an affinity-based mass spectrometric method in which proteins of interest are selectively absorbed to a chemically modified surface on a biochip. This technology may provide protein profiling of a variety of biological specimens. In this study, we explored whether the protein biochip SELDI approach could differentiate cervical cancer from non-cancer cohorts. We screened protein profiles generated by SELDI in 62 cervical epithelial cell samples microdissected from 35 invasive cervical cancer and 27 age-matched normal cervix tissue specimens, respectively. The cell lysates of pure populations of cervical cells were applied onto Ciphergen ProteinChip WCX2 Arrays. Proteins bound to the chips were analyzed on a ProteinChip Reader Model PBS II. Derived proteomic patterns were converted to a simple proteomic scoring for distinguishing cancer from non-cancer cohorts. SELDI protein profiles of cell lysates from 20 cervical cancer and 15 normal cervix tissue specimens were used to train and develop a classification scoring system that used a seven-protein mass pattern. The training samples could be correctly discriminated. When a test set of 27 samples was used for evaluation of this scoring system to distinguish cervical cancer from non-cancer, a sensitivity of 87%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 86% for the test population were obtained. All seven proteins appeared to be down regulated in cervical cancer. The results from this study indicate that the proteomics approach of SELDI mass spectrometry, in combination with a simple scoring system, may distinguish cervical cancer from its normal counterpart. If this approach is also workable in the analysis of cervical exfoliated cell lysate, it might potentially be used in the early diagnosis of invasive cervical cancer. In addition, the identification of these specific proteins in cervical cancer may also facilitate the discovery of new cervical tumor marker(s).

Adult↗
PubMed2006

Quantitative proteomic and genomic profiling reveals metastasis-related protein expression patterns in gastric cancer cells.

Gastric cancer is a leading cause of death worldwide, and patients have an overall 5-year survival rate of less than 10%. Using quantitative proteomic techniques together with microarray chips, we have established comprehensive proteome and transcriptome profiles of the metastatic gastric cancer TMC-1 cells and the noninvasive gastric cancer SC-M1 cell. Our qualitative protein profiling strategy offers the first comprehensive analysis of the gastric cancer cell proteome, identifying 926 and 909 proteins from SC-M1 and TMC-1 cells, respectively. Cleavable isotope-coded affinity tagging analysis allows quantitation of a total of 559 proteins (with a protein false-positive rate of <0.005), and 240 proteins were differentially expressed (>1.3-fold) between the SC-M1 and TMC-1 cells. We identified numerous proteins not previously associated with gastric cancer. Notably, a large subset of differentially expressed proteins was associated with tumor metastasis, including proteins functioning in cell-cell and cell-extracellular matrix (cell-ECM) adhesion, cell motility, proliferation, and tumor immunity. Gene expression profiling by DNA microarray revealed differential expression (of >2-fold) of about 1000 genes. The weak correlation observed between protein and mRNA profiles highlights the important complementarities of DNA microarray and proteomics approaches. These comparative data enabled us to map the disease-perturbed cell-cell and cell-ECM adhesion and Rho GTPase-mediated cytoskeletal pathways. Further validation of a subset of genes suggests the potential use of vimentin and galectin 1 as markers for metastasis. We demonstrate that combining proteomic and genomic approaches not only provides a rapid, robust, and sensitive platform to elucidate the molecular mechanisms underlying gastric cancer metastasis but also may identify candidate diagnostic markers and therapeutic targets.

Biomarkers, Tumor↗
PubMed2003

Multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome.

In order to discover novel protein markers indicative of disease processes or drug effects, the proteomics technology platform most commonly used consists of high resolution protein separation by two-dimensional electrophoresis (2-DE), mass spectrometric identification of proteins from stained gel spots and a bioinformatic data analysis process supported by statistics. This approach has been more successful in profiling proteins and their disease- or treatment-related quantitative changes in tissue homogenates than in plasma samples. Plasma protein display and quantitation suffer from several disadvantages: very high abundance of a few proteins; high heterogeneity of many proteins resulting in long charge trains; crowding of 2-DE separated protein spots in the molecular mass range between 45-80 kD and in the isoelectric point range between 4.5 and 6. Therefore, proteomic technologies are needed that address these problems and particularly allow accurate quantitation of a larger number of less abundant proteins in plasma and other body fluids. The immunoaffinity-based protein subtraction chromatography (IASC) described here removes multiple proteins present in plasma and serum in high concentrations effectively and reproducibly. Applying IASC as an upfront plasma sample preparation process for 2-DE, the protein spot pattern observed in gels changes dramatically and at least 350 additional lower abundance proteins are visualized. Affinity-purified polyclonal antibodies (pAbs) are the immunoaffinity reagents used to specifically remove the abundant proteins such as albumin, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, alpha-1-antitrypsin, hemopexin, transthyretin, alpha-2-HS glycoprotein, alpha-1-acid glycoprotein, alpha-2-macroglobulin and fibrinogen from human plasma samples. To render the immunoaffinity subtraction procedure recyclable, the pAbs are immobilized and cross-linked on chromatographic matrices. Antibody-coupled matrices specific for one protein each can be pooled to form mixed-bed IASC columns. We show that up to ten affinity-bound plasma proteins with similar solubility characteristics are eluted from a mixed-bed column in one step. This facilitates automated chromatographic processing of plasma samples in high throughput, which is desirable in proteomic disease marker discovery projects.

Antibodies↗
PubMed2003

Proteome analysis by mass spectrometry.

The coupling of high-performance mass spectrometry instrumentation with highly efficient chromatographic and electrophoretic separations has enabled rapid qualitative and quantitative analysis of thousands of proteins from minute samples of biological materials. Here, we review recent progress in the development and application of mass spectrometry-based techniques for the qualitative and quantitative analysis of global proteome samples derived from whole cells, tissues, or organisms. Techniques such as multidimensional peptide and protein separations coupled with mass spectrometry, accurate mass measurement of peptides from global proteome digests, and mass spectrometric characterization of intact proteins hold great promise for characterization of highly complex protein mixtures. Advances in chemical tagging and isotope labeling techniques have enabled quantitative analysis of proteomes, and highly specific isolation strategies have been developed aimed at selective analysis of posttranslationally modified proteins.

Chromatography, Affinity↗
PubMed2002

Identification of novel immunogenic proteins of Helicobacter pylori by proteome technology.

Cell surface proteins of the human gastric pathogen Helicobacter pylori, reference strain CCUG 17874, were extracted with acid glycine and fractionated by heparin affinity chromatography. The extracts were subsequently analysed using high-resolution two-dimensional gel electrophoresis (2-DE) and immunoblotting. Four proteins of low molecular masses (25-30 kDa) stained by Coomassie R-350, were identified by peptide ESI-MS/MS sequencing after in-gel tryptic digestion. The identified proteins were recognised by sera from H. pylori-infected patients. Two of them are now described for the first time as immunogenic proteins of which one protein was determined to be distinct from all H. pylori proteins previously described. In addition, the specificity of the identified peptides was evaluated using both 1-D and 2-D immunoblotting against a panel of sera from patients with various bacterial infections. The present identification of highly specific antigens of H. pylori will encourage the improvement of serological diagnostic tests to diagnose and monitor H. pylori infection.

Antigens, Bacterial↗
PubMed2004

Thioredoxin targets of developing wheat seeds identified by complementary proteomic approaches.

The role of thioredoxin in wheat starchy endosperm was investigated utilizing two proteomic approaches. Thioredoxin targets were isolated from total KCl-soluble extracts of endosperm and flour and separated by 2-DE following (1) reduction of the extract by the NADP/thioredoxin system and labeling the newly generated sulfhydryl (SH) groups with monobromobimane (mBBr), and, in parallel, (2) trapping covalently interacting proteins on an affinity column prepared with mutant thioredoxin h in which one of the active site cysteines was replaced by serine. The two procedures were complementary: of the total targets, one-third were observed with both procedures and one-third were unique to each. Altogether 68 potential targets were identified; almost all containing conserved cysteines. In addition to confirming known interacting proteins, we identified 40 potential thioredoxin targets not previously described in seeds. A comparison of the results obtained with young endosperm (isolated 10 days after flowering) to those with mature endosperm (isolated 36 days after flowering) revealed a unique set of proteins functional in processes characteristic of each developmental stage. Flour contained 36 thioredoxin targets, most of which have been found in the isolated developing endosperm.

Bridged Bicyclo Compounds↗
PubMed2002

A proteomic approach for the identification of cell-surface proteins shed by metalloproteases.

Proteolytic cleavage (shedding) of extracellular domains of many membrane proteins by metalloproteases is an important regulatory mechanism used by mammalian cells in response to environmental and physiological changes. Here we describe a proteomic system for analyzing cell surface shedding. The method utilized short-term culture supernatants from induced cells as starting material, followed by lectin-affinity purification, deglycosylation, and polyacrylamide gel electrophoresis separation. Relative quantitation of proteins was achieved via isotope dilution. In this study, a number of proteins already known to be shed were identified from activated monocytes and endothelial cells, thereby validating the method. In addition, a group of proteins were newly identified as being shed. The method provides an unbiased means to screen for shed proteins.

Adult↗
PubMed2005

Plasma membrane-focused proteomics: dramatic changes in surface expression during the maturation of human dendritic cells.

The differential expression of surface molecules on dendritic cells (DC) reflects their functional differences as immature and mature subsets. It is difficult, however, to characterize differences in surface expression by standard proteomic approaches, due mainly to the hydrophobic nature and low abundance of the individual proteins in question. We have established a method for obtaining high-yield plasmalemma preparations which contain surface molecules enriched more than 200-fold by coating cells with beads conjugated with antibody against a cell type-specific cell-surface molecule, followed by nitrogen cavitated disruption, magnetic separation, and density gradient ultracentrifugation. We identified and quantified 339 human monocyte-derived DC transmembrane proteins, including 33 previously uncharacterized molecules. Whereas 106 proteins were selectively expressed in immature cells or down-regulated after maturation, 191 proteins were selectively expressed in mature cells or up-regulated after maturation.

Cell Membrane↗