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1.
In the 1990s, a technique was developed to transfer proteins from electrophoresis gels onto poly(vinylidene difluoride) (PVDF) membranes, digest the proteins on the membranes with proteases such as trypsin and analyze the resulting peptides on the membranes directly by mass spectrometry to identify the proteins. This technique, based on gel electrophoresis, is particularly useful for analyzing protein isoforms, splicing variants and post-translationally modified proteins. Previously, the low ionization efficiency of peptides immobilized on the membranes often rendered this technique useless. However, this technique has been improved by the use of PVDF membranes with a small pore size, which has enabled highly efficient and effective electroblotting and mass spectrometric analyses. Here, the advantage of this technique is discussed. 相似文献
3.
In Photosystem II (PSII), a high number of plastid encoded and membrane integral low molecular weight proteins smaller than 10 kDa, the proteins PsbE, F, H, I, J, K, L, M, N, Tc, Z and the nuclear encoded PsbW, X, Y1, Y2 proteins have been described. Here we show that all low molecular weight proteins of PSII already accumulate in the etioplast membrane fraction in darkness, whereas PsaI and PsaJ of photosystem I (PSI) represent the only low molecular weight proteins that do not accumulate in darkness. We found by BN‐PAGE separation of membrane protein complexes and selective MS that the accumulation of one‐helix proteins from PSII is light independent and occurs in etioplasts. In contrast, in chloroplasts isolated from light‐grown plants, low molecular weight proteins were found to specifically accumulate in PSI and II complexes. Our results demonstrate how plants grown in darkness prepare for the induction of chlorophyll dependent photosystem assembly upon light perception. We anticipate that our investigation will provide the essential means for the analysis of protein assembly in any membrane utilizing low molecular weight protein subunits. 相似文献
4.
Introduction: Viral diseases contribute much to human and animal suffering and enormous efforts are directed at developing appropriate vaccines for protection. Glycoproteins constitute much of the viral surfaces and are obvious targets for such vaccine development. This review describes mass spectrometric methods used for the structural determination of these compounds. Areas covered: The review describes the structures of the N- and O-linked glycans found on glycoproteins and mass spectrometric methods for their ionization and fragmentation. The steps, such as determination of glycan attachment sites and the structures of the attached glycans following their release from the glycoproteins are described and examples are given of the uses of the various analytical methods using mainly influenza, Ebola and HIV as representative examples. Also included are tables listing work on many other viruses. Expert commentary: Recent technological advances, such as the introduction of ion mobility techniques, have greatly improved analyses in this area and have enabled larger amounts of information to be gathered in shorter time periods on ever smaller amounts of material. Such techniques should greatly accelerate the discovery of vaccine targets and lead to the production of vaccines for diseases not currently available. 相似文献
5.
Mitochondrial membrane permeabilization is a rate-limiting step of cell death. This process is, at least in part, mediated by opening of the permeability transition pore complex (PTPC) Several soluble proteins from the mitochondrial intermembrane space and matrix are involved in the activation of catabolic hydrolases including caspases and nucleases. We therefore investigated the composition of a mixture of proteins released from purified mitochondria upon PTPC opening. This mixture was subjected to a novel proteomics/mass spectrometric approach designed to identify a maximum of peptides. Peptides from a total of 79 known proteins or genes were identified. In addition, 21 matches with expressed sequence tags (EST) were obtained. Among the known proteins, several may have indirect or direct pro-apoptotic properties. Thus endozepine, a ligand of the peripheral benzodiazepin receptor (whose occupation may facilitate mitochondrial membrane permeabilization), was found among the released proteins. Several proteins involved in protein import were also released, namely the so-called X-linked deafness dystonia protein (DDP) and the glucose regulated protein 75 (grb75), meaning that protein import may become irreversibly disrupted in mitochondria of apoptotic cells. In addition, a number of catabolic enzymes are detected: arginase 1 (which degrades arginine), sulfite oxidase (which degrades sulfur amino acids), and epoxide hydrolase. Although the functional impact of each of these proteins on apoptosis remains elusive, the present data bank of mitochondrial proteins released upon PTPC opening should help further elucidation of the death process. 相似文献
6.
Electrophoretic mobility shift assays (EMSA) are commonly employed for the analysis of nucleic acid/ protein interactions with a native gel system. Here, we report a method to identify RNA binding proteins from a dried EMSA gel by mass spectrometry following autoradiography. Compared to wet gel exposure, our approach resulted in an improved protein identification sensitivity and RNA/protein complex isolation accuracy. The method described here is useful for the large scale characterization of RNA- or DNA-protein complexes. 相似文献
7.
The entire genomic DNA sequences of a number of prokaryotic and eukaryotic species are now available and many more, including the human genome, will be completed in the near future. The state-of-life of a cell at any given time, however, is defined by its protein composition, i.e., its proteome. Gel electrophoresis, mass spectrometry, and bioinformatics will be important tools for protein and proteome analysis in the post-genome era. Protein identification from electrophoretic gels by mass spectrometric peptide mapping or peptide sequencing combined with sequence database searching is established and has been applied to numerous biological systems. We describe current strategies and selected applications in molecular and cell biology. The next challenges are detailed structure/function analyses, which include studying the molecular composition of multiprotein complexes and characterization of secondary modifications of proteins. The advantages and limitations of a number of mass spectrometry-based strategies designed for microcharacterization of low amounts of protein from electrophoretic gels are discussed and illustrated by examples. Proteins Suppl. 2:74–89, 1998. © 1998 Wiley-Liss, Inc. 相似文献
8.
It has long been thought that blood plasma could serve as a window into the state of one's organs in health and disease because tissue-derived proteins represent a significant fraction of the plasma proteome. Although substantial technical progress has been made toward the goal of comprehensively analyzing the blood plasma proteome, the basic assumption that proteins derived from a variety of tissues could indeed be detectable in plasma using current proteomics technologies has not been rigorously tested. Here we provide evidence that such tissue-derived proteins are both present and detectable in plasma via direct mass spectrometric analysis of captured glycopeptides and thus provide a conceptual basis for plasma protein biomarker discovery and analysis. 相似文献
9.
The modification of H3 in asynchronous HeLa cells was profiled using Top Down Mass Spectrometry. A broad distribution of species differing by 14 Da and containing less than 3% unmodified protein was observed for all three variants. Species of up to +168 Da were observed for H3.1, and fragmentation of all species by Electron Capture Dissociation (ECD) revealed approximately 5% methylation of K4 and approximately 50% dimethylation of K9. K14 and K23 were major sites of acetylation. H3.3 was slightly hypermodified with the apex of the distribution shifted by approximately +14 Da compared to H3.1. H3.1 (50% and 15%) from colchicine-treated cells was monophosphorylated and diphosphorylated, respectively, with equivalent modification of S10 and S28. 相似文献
10.
Electrospray ionization-mass spectrometry of ion-exchange and reverse-phase purified hemocyanin from Limulus polyphemus revealed six predominant isoforms with molecular weights ranging from 71,730 to 72,695 Da. The heaviest of these agreed closely with the molecular weight calculated for the previously determined Edman sequence with substitution of isoleucine for valine at position 9 and inclusion of three internal disulfide bonds and one copper atom. Proposed structures for the other isoforms were made on the basis of the molecular weight measurements. Reverse-phase chromatography can be used in addition to the traditional ion-exchange step to produce hemocyanin preparations of greater purity that might be valuable for further detailed investigations of the physicochemical properties of these important proteins. The results reflect yet again the value of mass spectrometry for recognizing molecular microheterogeneity in biological macromolecules and for following protein purification. 相似文献
11.
N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme that primarily degrades palmitoylethanolamine (PEA), a lipid amide that inhibits inflammatory responses. We developed a HEK293 cell line stably expressing the NAAA pro-enzyme (zymogen) and a single step chromatographic purification of the protein from the media. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry MALDI-TOF MS analysis of the zymogen (47.7 kDa) treated with peptide-N-glycosidase F (PNGase F) identified 4 glycosylation sites, and acid cleavage of the zymogen into α- and β-subunits (14.6 and 33.3 kDa) activated the enzyme. Size exclusion chromatography estimated the mass of the active enzyme as 45 ± 3 kDa, suggesting formation of an α/β heterodimer. MALDI-TOF MS fingerprinting covered more than 80% of the amino acid sequence, including the N-terminal peptides, and evidence for the lack of a disulfide bond between subunits. The significance of the cysteine residues was established by their selective alkylation resulting in almost complete loss of activity. The purified enzyme was kinetically characterized with PEA and a novel fluorogenic substrate, N-(4-methyl coumarin) palmitamide (PAMCA). The production of sufficient quantities of NAAA and a high throughput assay could be useful in discovering novel inhibitors and determining the structure and function of this enzyme. 相似文献
12.
The sarcoplasmic reticulum from skeletal muscle constitutes an elaborate membrane system that contains a considerable number of integral and very large proteins that exist in highly complex supramolecular clusters. Conventional proteomics using two-dimensional gel electrophoresis greatly underestimates the presence of these proteins. Here, we have applied one-dimensional gradient gels and on-membrane digestion to overcome this technical problem. Mass spectrometric analysis has determined the presence of 31 distinct protein species in the sarcoplasmic reticulum, including key Ca2+-handling proteins such as the ryanodine receptor, Ca2+-ATPase, calsequestrin and sarcalumenin. Immunoblotting confirmed the relative position of these Ca2+-regulatory elements in analytical gel replicas. Interestingly, aldolase and phosphofructokinase were found to be present in the purified sarcoplasmic reticulum, supporting the idea of a close physical coupling between the glycolytic pathway and the energy-dependent sarcoplasmic reticulum. Hence, on-membrane digestion is highly suitable as the method of choice for studying integral and high-molecular-mass proteins in proteomic studies. 相似文献
13.
The cDNA coding for amino acid residues 121-269 of human interleukin 1 beta was cloned and expressed at Sclavo Research Center by C. Baldari et al. (1987, EMBO J. 6, 229-234) using Saccharomyces cerevisiae as the host cell. The purified protein showed a very low specific activity when compared to that of a mature recombinant interleukin expressed from Escherichia coli. Preliminary experiments indicated the occurrence of post-translational events in the yeast-derived protein. In an attempt to correlate the structural modifications and the biological activity of recombinant interleukins, the two proteins were characterized by fast atom bombardment mass spectrometry (FAB/MS) following the FAB-mapping procedure. The amino acid sequence of interleukin expressed in E. coli was identical to that expected whereas the mass spectrometric analysis of the recombinant S. cerevisiae protein confirmed the occurrence of covalent modifications. In particular, the asparagine residue at position 7 (numbering follows the mature active protein sequence) was shown to be glycosylated and the two cysteine residues at position 8 and 71 were involved in an S-S bridge. The results demonstrate the value of FAB/MS in the quality control of recombinant interleukins. 相似文献
14.
The 26S proteasome is a multisubunit complex responsible for degradation of ubiquitinated substrates, which plays a critical role in regulating various biological processes. To fully understand the function and regulation of the proteasome complex, an important step is to elucidate its subunit composition and posttranslational modifications. Toward this goal, a new affinity purification strategy has been developed using a derivative of the HB tag for rapid isolation of the human 26S proteasome complex for subsequent proteomic analysis. The purification of the complex is achieved from stable 293 cell lines expressing a HB-tagged proteasome subunit and by high-affinity streptavidin binding with TEV cleavage elution. The complete composition of the 26S proteasome complex, including recently assigned new subunits, is identified by LC-MS/MS. In addition, all known proteasome activator proteins and components involved in the ubiquitin-proteasome degradation pathway are identified. Aside from the subunit composition, the N-terminal modification and phosphorylation of the proteasome subunits have been characterized. Twelve novel phosphorylation sites from eight subunits have been identified, and N-terminal modifications are determined for 25 subunits, 12 of which have not been previously reported in mammals. We also observe different N-terminal processing of subunit Rpn2, which results in identification of two different N-termini of the protein. This work presents the first comprehensive characterization of the human 26S proteasome complex by affinity purification and tandem mass spectrometry. The detailed proteomic profiling obtained here is significant to future studies aiming at a complete understanding of the structure-function relationship of the human 26S proteasome complex. 相似文献
15.
The trisaccharide present in the kaempferol 3-triglucoside-7-rhamnoside of potato seed has been identified by hydrolytic experiments and by MS measurements on the perdeuteriomethylated glycoside as sophorotriose (glucosyl-β1 → 2-glucosyl-β1 → 2-glucose). 相似文献
17.
The stomatogastric ganglion (STG) and the cardiac ganglion (CG) of decapod crustaceans are modulated by neuroactive substances released locally and by circulating hormones released from neuroendocrine structures including the pericardial organs (POs). Using nanoscale liquid chromatography electrospray ionization quadrupole-time-of-flight tandem mass spectrometry and direct tissue matrix-assisted laser desorption/ionization Fourier transform mass spectrometry we have identified and sequenced a novel neuropeptide, GAHKNYLRFamide (previously misassigned as KHKNYLRFamide in a study that did not employ peptide derivatization), from the POs and/or the stomatogastric nervous system (STNS) of the crabs, Cancer borealis, Cancer productus and Cancer magister. In C. borealis, exogenous application of GAHKNYLRFamide increased the burst frequency and number of spikes per burst of the isolated CG and re-initiated bursting activity in non-bursting ganglia, effects also elicited by the FMRFamide-like peptides (FLPs) SDRNFLRFamide and TNRNFLRFamide. In the intact STNS (which contains the STG), exogenous application of GAHKNYLRFamide increased the frequency of the pyloric rhythm and activated the gastric mill rhythm, effects also similar to those elicited by SDRNFLRFamide and TNRNFLRFamide. FLP-like immunoreactivity in the POs and the STNS was abolished by pre-adsorption with the synthetic GAHKNYLRFamide. Different members of the FLP family exhibited differential degradation in the presence of extracellular peptidases. Taken collectively, the amino acid sequence of GAHKNYLRFamide, the blocking of FLP-like immunostaining, and its physiological effects on the CG and STNS suggest that this peptide is a novel member of the FLP superfamily. 相似文献
18.
Heparin like glycosaminoglycans (HLGAGs) are struc-turally complex linear polysaccharides composed of re-peating disaccharide unit of uronic (α-L-iduronic or β-D-glucuronic) acid linked 1→4 to α-D-glucosamine, whichis a highly variable sulfation pattern and ascribes to eachglycosaminoglycan (GAG) chain a unique structuralsignature. This signature dictates specific the GAG-pro-tein interactions underlying critical biological processesrelated to cell and tissue functions [1]. Only in fe… 相似文献
19.
Integral membrane proteins are among the most interesting molecules for biomedical research, as some of the most important cellular functions are inherently tied to biological membranes. One such example is the vast expanse of receptors on cell surfaces. However, due to difficulties in the biochemical purification and structure/function analysis of membrane proteins, caused by their hydrophobic or amphophilic nature, membrane proteins are still much less studied than soluble proteins. Our laboratory has successfully developed and applied a methodology for the mass spectrometric analysis of integral membrane proteins. Here, we present an improvement in the sensitivity of detection made possible by the advancement of mass spectrometric instrumentation and refinement of the chromatographic analysis. Subpicomolar samples of bovine rhodopsin purified from native membranes were successfully analyzed, obtaining complete sequence coverage and the detection and localization of posttranslational modifications. Therefore, it is demonstrated that the detection limits and sequence coverage for soluble and membrane proteins can be comparable. The methodology presented here allows mass spectrometric analysis of subpicomolar levels of photopigments or other integral membrane proteins either from their native membranes or as products of expression systems. 相似文献
20.
In the present study, we have used a combination of 2-DE and MS to isolate and characterize two variants of the mitochondrial complex I subunit NDUFA10 from Wistar rat brain. Extensive MS/MS analysis revealed that a D/N substitution at position 120 resulting from a 353A/G transition in the coding gene is the biochemical difference between the two most abundant NDUFA10 isoforms. Moreover, 33 modifications of distinct chemical nature targeting 59 specific residues were found to be common to the acidic and basic forms. Positions C67, H149 and H322 of NDUFA10 were specially targeted by different modifications suggesting the high reactivity of these residues and their potential implication in the regulation of the protein function. Together with nonenzymatic modifications that can form in the sample isolation and workup steps, such as oxidation of methionine, tryptophan, cysteine and histidine, we describe amino acid variants of unknown chemical structure that must be further characterized, as well as accumulation of R, K and H methylations and probably K acetylations at the C-terminal region that might play a role in the control of NDUFA10 activity according to similar mechanisms to those described for histones. 相似文献
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