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1.
Delineating protein-protein interactions via biomolecular interaction analysis-mass spectrometry 总被引:3,自引:0,他引:3
The utility of biomolecular interaction analysis-mass spectrometry (BIA/MS) in screening for protein-protein interactions was explored in this work. Experiments were performed in which proteins served as ligands for screening of possible interactions with other proteins from human plasma and urine. The proteins utilized were beta-2-microglobulin, cystatin C (cysC), retinol binding protein (RBP), transthyretin (TTR), alpha-1-microglobulin, C-reactive protein, transferrin and papain. The immobilization of functionally active proteins was confirmed via interactions with antibodies to the corresponding proteins. Various dilutions of human urine and plasma were injected over the protein-derivatized surfaces. It was observed that the urine injections generally yielded smaller SPR responses than those observed after the plasma injections. The BIA/MS experiments did not reveal novel protein-protein interactions, although several established interactions (such as those between RBP and TTR, and cysC and papain) were validated. Few protein ligand deficiencies (such as truncations) leading to false negative and false positive BIA/MS results were also discovered. 相似文献
2.
Ongoing, worldwide efforts in genomic and protein sequencing, and the ability to readily access corresponding sequence databases, have emphatically driven the development of high‐performance bioanalytical instrumentation capable of characterizing proteins and protein–ligand interactions with great accuracy, speed and sensitivity. Two such analytical techniques have arisen over the past decade to play key roles in the characterization of proteins: surface plasmon resonance biomolecular interaction analysis (SPR‐BIA) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF). SPR‐BIA is used in the real‐time investigation of biomolecular recognition events, and is thereby capable of providing details on the association and dissociation kinetics involved in the interaction, information ultimately leading to the determination of dissociation constants involved in the event. MALDI‐TOF is used in the structural characterization, identification and sensitive detection of biomolecules. Although the two techniques have found many independent uses in bioanalytical chemistry, the combination of the two, to form biomolecular interaction analysis mass spectrometry (BIA/MS), enables a technique of analytical capabilities greater than those of the component parts. Reviewed here are issues of concern critical to maintaining high‐levels of performance throughout the multiplexed analysis, as well as examples illustrating the potential analytical capabilities of BIA/MS. Copyright © 1999 John Wiley & Sons, Ltd. 相似文献
3.
Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional analytical technique that quantitatively and qualitatively detects analytes of interests. In the first dimension, surface plasmon resonance (SPR) is utilized for detection of biomolecules in their native environment. Because SPR detection is non-destructive, analyte(s) retained on the SPR-active sensor surface can be analyzed in a second dimension using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The qualitative nature of the MALDI-TOF MS analysis complements the quantitative character of SPR sensing and overcomes the shortcomings of the SPR detection stemming from the inability to differentiate and characterize multi-protein complexes and non-specific binding. In this work, the benefit of performing MS analysis following SPR sensing is established. Retrieval and detection of four markers present in biological fluids (cystatin C, beta-2-microglobulin, urinary protein 1 and retinol binding protein) was explored to demonstrate the effectiveness of BIA/MS in simultaneous detection of clinically related biomarkers and delineation of non-specific binding. Furthermore, the BIA/MS limit of detection at very low SPR responses was investigated. Finally, detection of in-vivo assembled protein complexes was achieved for the first time using BIA/MS. 相似文献
4.
Surface plasmon resonance and mass spectrometry (SPR-MS) has been combined for quality check of recombinant 6xHis-tagged 14-3-3
proteins expressed in Escherichia coli. Lysates were injected over an SPR sensorchip with immobilized Ni2+ for SPR analysis of the specific Ni2+ binding response and stability. To validate the identity, intactness and homogeneity of the captured proteins were eluted
for mass spectrometric analysis of intact molecular weight and peptide mass mapping. Additionally, the captured recombinant
proteins were investigated for specific binding to known phosphorylated ligands of 14-3-3 proteins in order to test their
activity.
Specific binding of recombinant and native 14-3-3 proteins in complex mixtures to immobilized phosphopeptides and subsequent
elution was also tested by SPR-MS. Ammonium sulfate precipitate fractions from lysates of E. coli expressing 14-3-3 protein and of cauliflower were investigated for specific binding to the phosphopeptide ligands immobilized
on a sensorchip by SPR. Subsequently, the bound protein was eluted and analyzed by MS for characterization of intact mass
and peptide mass mapping. 相似文献
5.
6.
Complex molecular changes associated with early stage human heart disease are poorly understood and prevent the development of effective treatments of human cardiac disease. Relatively minor structural changes in early disease may accompany some conditions such as arrhythmias. Our objective was to determine if significant proteomic changes occur in heart tissues in the absence of structural pathology. We used a proteomic "pipeline" based on Ciphergen SELDI-TOF/MS, gel electrophoresis and MALDI-TOF/MS. The kyphoscoliosis (ky) mouse carries a mutation in a putative transglutaminase causing a primary skeletal muscle disease. The ky protein is expressed usually in skeletal and cardiac muscle but its absence from the ky heart causes no structural pathology making it a good model of "occult" heart disease. We discovered 20 statistically validated biomarkers discriminating ky from normal hearts, one cardiac troponin-I was reduced by 40% in ky hearts. A 17% deficit was confirmed subsequently by Western blot. Thus, the proteome of ky hearts was abnormal, giving support to our contention that this SELDI-based analytical approach is capable of making a significant contribution to the analysis of complex proteomic changes in early stage human heart disease. 相似文献
7.
Shi Y Shen B Xiang P Yan H Shen M 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(30):3161-3166
Ethyl glucuronide (EtG) has been shown to be a suitable marker of excessive alcohol consumption. Determination of EtG in hair samples may help to differentiate social drinkers from alcoholics, and this testing can be widely used in forensic science, treatment programs, workplaces, military bases as well as driving ability test to provide legal proof of drinking. A method for determination of EtG in hair samples using large volume injection-gas chromatography-tandem mass spectrometry (LVI-GC/MS/MS) was developed and validated. Hair samples (in 1 mL deionized water) were ultrasonicated for 1h and incubated overnight; these samples were then deproteinated to remove impurities and derivatisated with 15 μL of pyridine and 30 μL of BSTFA. EtG was detected using GC/MS/MS in multiple-reaction monitoring mode. This method exhibited good linearity: y=0.0036 x+0.0437, R2=0.9993, the limit of detection and the limit of quantification were 5 pg/mg and 10 pg/mg, respectively. The extraction recoveries were more than 60%, and the inter-day and intra-day relative standard deviations (RSD) were less than 15%. This method has been applied to the analysis of EtG in hair samples from 21 Chinese subjects. The results for samples obtained from all of those who were teetotallers were negative, and the results for the other 15 samples ranged from 10 to 78 pg/mg, except for one negative sample. These data are the basis for interpretation of alcohol abuse. 相似文献
8.
We report the first use of functionalized cadmium selinide quantum dots (CdSe QDs) with 11‐mercaptoundecanoic acid (MUA) as the matrix for the selective ionization of proteins with high resolution and rapid analysis of amino acids and peptides by using quantum dots laser desorption/ionization mass spectrometry (QDLDI‐MS). The mercaptocarboxylic groups of CdSe QDs have been known to be an effective affinity probe to interact with the biomolecules at low abundance level. Using these QDs as the matrix, sensitivity of the method was greatly enhanced and the LOQ of peptides was found to be 100 pM with RSD <10%. The QDLDI‐MS is capable for the selective ionization of insulin, lysozyme and myoglobin with high resolution, which is not observed with sinapic acid (SA) as the matrix. The QDLDI‐MS technique offers many advantages for the analysis of amino acids, peptides and proteins with regard to simplicity, rapidity, sensitivity and the mass spectra were generated in the presence of signal suppressors such as urea and Trition X‐100. In addition, the CdSe QDs have been successfully applied as preconcentrating probes for the analysis of digested peptides in lysozyme from chicken egg white by microwave‐assisted enzymatic digestion. This indicates that the QDs are able to absorb radiation from microwave and their ability to trap peptides from microwave‐digested lysozyme. These results demonstrate that the CdSe QDs are promising candidates for the selective ionization of the analytes with an accurate platform to the rapid screening of biomolecules. 相似文献
9.
The tau protein plays an important role in some neurodegenerative diseases including Alzheimer's disease (AD). Neurofibrillary tangles (NFTs), a biological marker for AD, are aggregates of bundles of paired helical filaments (PHFs). In general, the alpha-sheet structure favors aberrant protein aggregates. However, some reports have shown that the alpha-helix structure is capable of triggering the formation of aberrant tau protein aggregates and PHFs have a high alpha-helix content. In addition, the third repeat fragment in the four-repeat microtubule-binding domain of the tau protein (residues 306-336: VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQ, according to the longest tau protein) adopts a helical structure in trifluoroethanol (TFE) and may be a self-assembly model in the tau protein. In the human brain, there is a very small quantity of copper, which performs an important function. In our study, by means of matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy, the binding properties of copper (II) ion to the R3 peptide derived from the third repeat fragment (residues 318-335: VTSKCGSLGNIHHKPGGG) have been investigated. The results show that copper ions bind to the R3 peptide. CD spectra, ultraviolet (UV)-visible absorption spectra, and MALDI-TOF MS show pH dependence and stoichiometry of Cu2+ binding. Furthermore, CD spectra and NMR spectroscopy elucidate the copper binding sites located in the R3 peptide. Finally, CD spectra reveal that the R3 peptide adopts a mixture structure of random structures, alpha-helices, and beta-turns in aqueous solutions at physiological pH. At pH 7.5, the addition of 0.25 mol eq of Cu2+ induces the conformational change from the mixture mentioned above to a monomeric helical structure, and a beta-sheet structure forms in the presence of 1 mol eq of Cu2+. As alpha-helix and beta-sheet structures are responsible for the formation of PHFs, it is hypothesized that Cu2+ is an inducer of self-assembly of the R3 peptide and makes the R3 peptide form a structure like PHF. Hence, it is postulated that Cu2+ plays an important role in the aggregation of the R3 peptide and tau protein and that copper (II) binding may be another possible involvement in AD. 相似文献
10.
For many insect species, pheromones are important communication tools, but chemical analysis and experimental study can be technically challenging because they require the detection and handling of complex chemicals in small quantities. One drawback of traditional mass spectrometry methods such as gas chromatography mass spectrometry is that whole‐body extractions from one to several hundred individuals are required, with the consequence that intra‐ and interindividual differences cannot be detected. Here, we used the recently introduced UV‐LDI MS (ultraviolet laser desorption/ionization mass spectrometry) to profile the ‘osmeterium’ of the sepsid fly Themira superba that is located on the edge of the hind tibia of males. Based on analyses of individual legs, we established that the gland produced a secretion that consisted of oxygenated hydrocarbons and putative isoprenoids. The secretion was first detected 24 h after eclosion, and its transfer to the wings of females during mating was demonstrated using UV‐LDI MS. We then tested whether the secretion had an anti‐aphrodisiac function, but experimental transfer of the secretion to virgin females did not affect mating success or copulation duration. Throughout the study, UV‐LDI MS proved invaluable, because it allowed tracking the natural and experimental transfer of small quantities of pheromones to specific body parts of small flies. 相似文献
11.
Natalicia de Jesus Antunes Lauro Wichert‐Ana Eduardo Barbosa Coelho Oscar Della Pasqua Veriano Alexandre Jr. Osvaldo Massaiti Takayanagui Eduardo Tozatto Vera Lucia Lanchote 《Chirality》2013,25(12):897-903
Oxcarbazepine is a second‐generation antiepileptic drug indicated as monotherapy or adjunctive therapy in the treatment of partial seizures or generalized tonic–clonic seizures in adults and children. It undergoes rapid presystemic reduction with formation of the active metabolite 10‐hydroxycarbazepine (MHD), which has a chiral center at position 10, with the enantiomers (S)‐(+)‐ and R‐(?)‐MHD showing similar antiepileptic effects. This study presents the development and validation of a method of sequential analysis of oxcarbazepine and MHD enantiomers in plasma using liquid chromatography with tandem mass spectrometry (LC‐MS/MS). Aliquots of 100 μL of plasma were extracted with a mixture of methyl tert‐butyl ether: dichloromethane (2:1). The separation of oxcarbazepine and the MHD enantiomers was obtained on a chiral phase Chiralcel OD‐H column, using a mixture of hexane:ethanol:isopropanol (80:15:5, v/v/v) as mobile phase at a flow rate of 1.3 mL/min with a split ratio of 1:5, and quantification was performed by LC‐MS/MS. The limit of quantification was 12.5 ng oxcarbazepine and 31.25 ng of each MHD enantiomer/mL of plasma. The method was applied in the study of kinetic disposition of oxcarbazepine and the MHD enantiomers in the steady state after oral administration of 300 mg/12 h oxcarbazepine in a healthy volunteer. The maximum plasma concentration of oxcarbazepine was 1.2 µg/mL at 0.75 h. The kinetic disposition of MHD is enantioselective, with a higher proportion of the S‐(+)‐MHD enantiomer compared to R‐(?)‐MHD and an AUC0‐12 S‐(+)/R‐(?) ratio of 5.44. Chirality 25:897–903, 2013. © 2013 Wiley Periodicals, Inc. 相似文献
12.
An efficient method to characterise complex plant extracts is described using the example of Piper methysticum Forst. (kava; Piperaceae). The method is based on the on-line coupling of high-performance liquid chromatography to a new detection technique: coordination ion spray-mass spectrometry (CIS/MS). CIS/MS is a universal, novel ionisation technique improving selectivity as well as sensitivity. Charged complexes were formed through addition of central complexing ions such as sodium, silver and cobalt. The advantages of CIS/MS detection compared with the electrospray ionisation detection are discussed. The experimental set-up and the application of this simple and robust technique is described to show the its various fields of application in the analysis of plant extracts. 相似文献
13.
Neuropeptide Y (NPY) in cerebrospinal fluid from patients with Huntington's Disease: increased NPY levels and differential degradation of the NPY1–30 fragment
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Leona Wagner Maria Björkqvist Sofia Hult Lundh Raik Wolf Arne Börgel Dagmar Schlenzig Hans‐Henning Ludwig Jens‐Ulrich Rahfeld Blair Leavitt Hans‐Ulrich Demuth Åsa Petersén Stephan von Hörsten 《Journal of neurochemistry》2016,137(5):820-837
14.
Neuropeptides are important with respect to almost all physiological processes and behavioural patterns in an organism. In the present study, muscle bioassays, immunohistochemistry and matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry are used to investigate the distribution and efficacy of proctolin in the antennal heart of 36 species of lower Neoptera. In total, 20 species of Dictyoptera (cockroaches, termites, praying mantids), eight species of Saltatoria (crickets and grasshoppers) and eight species of Phasmatodea (stick insects) are investigated. The antennal heart of all tested Blattoidea, including the termite Mastotermes darwiniensis Froggatt, exhibit a strong proctolin‐like immunoreactivity, as well as a high sensitivity to proctolin, whereas members of the second clade of cockroaches (Blaberoidea) are largely insensitive to proctolin and show only a weak proctolin‐like immunoreactivity. The antennal heart of praying mantids (Mantodea) also contains only few proctolin‐like immunoreactive fibres but is highly sensitive to proctolin. Such a high sensitivity is found also in Phasmatodea, although the antennal heart of these insects does not have proctolin‐like immunoreactive fibres. These findings are supported by MALDI‐TOF mass spectrometry; no trace of proctolin is detected in antennal heart preparations of Phasmatodea. In Saltatoria, only weak (or no) effects of proctolin are observed and, in most subtaxa, no proctolin‐like immunoreactivity is visible in the antennal heart preparations. Only in Grylloidea is strong proctolin‐like immunostaining found in processes of the antennal heart. In these species, weak or moderate effects of proctolin are observed in the antennal heart bioassay. In general, the differences in distribution of proctolin and effects on the antennal heart within the basal Neoptera cannot be deduced from their phylogenetic position, although they show conformity within each (sub)taxon. 相似文献
15.
《Bioscience, biotechnology, and biochemistry》2013,77(8):2184-2190
A sensitive and simple liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the detection of alginate oligosaccharides (AOs) in mouse plasma and urine after oral administration. In an AO mixture, dimer, trimer, and tetramer were detected by LC-MS/MS equipped with an anion-exchange column with extremely high sensitivity. By this method, we detected certain levels of AOs in samples prepared from mouse plasma and urine after a single oral administration of the AO mixture. Based on a calibration curve made with an AO trimer peak area as a standard, the maximum plasma and urine concentrations of AOs were estimated to be 24.5 μg/ml at 5 min and 425.5 μg/ml at 30 min, respectively. These results suggest that the LC-MS/MS method is well suited to pharmacokinetic analysis of AOs in an in vivo system, and that some of orally administered AOs, at least from dimer to tetramer, are absorbed by digestive organs promptly, and that unaltered, these oligomers were excreted into an urine after a single oral administration to a mouse. 相似文献
16.
《Expert review of proteomics》2013,10(6):815-829
Viruses constantly adapt to and modulate the host environment during replication and propagation. Both DNA and RNA viruses encode multifunctional proteins that interact with and modify host cell proteins. While viral genomes were the first complete sequences known, the corresponding proteomes are only now elucidated, with some surprising results. Even more daunting is the task to globally monitor the impact of viral infection on the proteome of the host cell and many technical hurdles must still be overcome in order to facilitate robust and reproducible measurements. Further complicating the picture is the dynamic nature of proteins, including post-translational modifications, enzymatic cleavage and activation or destruction by proteolytic events. Nevertheless, several promising studies have been published using high-throughput methods directly measuring protein abundance. Particularly, quantitative or semiquantitative mass spectrometry-based analysis of viral and cellular proteomes are now being used to characterize viruses and their host interaction. In addition, the full set of interactions between viral and host proteins, the interactome, is beginning to emerge, with often unexpected interactions that need to be carefully validated. In this review, we will discuss two major areas of viral proteomics: first, virion proteomics (such as the protein characterization of viral particles) and second, proteoviromics, including the viral protein interactomics and the quantitative analysis of host cell proteome during viral infection. 相似文献
17.
Introduction – The increasing demands of roots and rhizomes of Salvia miltiorrhiza almost exhausted the wild Salvia sources in China. However, the content and composition of phenolic acids in the aerial parts of the plant and their potential to be used as a substitute has not been explored. Objective – To evaluate the potential of the aerial parts of Salvia miltiorrhiza as new natural sources of phenolic acids. Methodology – HPLC coupled with diode array detection (DAD) and electrospray ionization multistage mass spectrometry (ESI/MSn) has been used for qualitative and quantitative analysis of phenolic compounds. Results – A total of 38 phenolic compounds were identified or tentatively characterized. A quantitative HPLC‐DAD method allowing the simultaneously quantification of six phenolic acids was optimized and validated for linearity, precision, accuracy, and limits of detection and quantification. Calibration curves showed good linear regression (r2 > 0.9991) within test ranges; the recoveries ranged between 95.64 and 101.67% and the RSDs were less than 3.01%. Conclusion – The developed methods have been proved to be effective for the identification and quantification of phenolic acids in S. miltiorrhiza. The results obtained suggest that the aerial parts of the plant could be used as an alternative source of sage phenolics. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
18.
Water-soluble complexes of the dietary carotenoid psi,psi-carotene (lycopene 1) with cyclomaltohexaose (alpha-cyclodextrin, alphaCD) and cyclomaltoheptaose (beta-cyclodextrin, betaCD) have been prepared and characterized via multiangle light scattering (MALS), ionspray/electrospray ionization (IS/ESI) mass spectrometry (MS) and tandem MS. MALS experiments point out that large aggregates of particles, on the nanometer-size scale, are present in water, with meaningful differences in the shape of the alphaCD/1 aggregates with respect to betaCD/1 analogues. The true 1:1 alphaCD/1 inclusion complex has been observed by IS/ESIMS and confirmed by tandem MS. The structure of CD/1 aggregations in water is proposed which are consistent with the combined MALS and MS experimental results. 相似文献
19.
Véronique Dubois Anne-Marie Lambeir Stefaan Vandamme Veerle Matheeussen Yves Guisez Simon Scharpé Ingrid De Meester 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(4):781-788
The dipeptidyl peptidases (DPP) 8 and 9 belong to the DPP4 activity and/or structure homologues (DASH). Recently, a DPP9-like protein was purified from bovine testes. The aim of the present study was to prove its identity and to investigate the characteristics of this natural enzyme. We report the identification and N-terminal sequence analysis by MALDI-TOF/TOF MS, of the purified bovine enzyme as DPP9. The tryptic peptides after in-gel digestion covered 41% and 38% of the short and full-length variants of bovine DPP9, respectively. Using Asp-N digestion combined with a very recently described mass spectrometric method using DITC glass beads, the N-terminal peptide (XTGALTSERG) was isolated. It corresponds to the N-terminus of the short form of bovine DPP9. There was no evidence for glycosylation of purified bovine DPP9. The purified DPP9 was activated and stabilized by DTT. Bovine DPP9 lost its activity almost completely after alkylation with N-ethylmaleimide. Also alkylation with iodoacetamide inhibited DPP9, albeit only 70%. Other properties of bovine DPP9 are reported, including functional stability and sensitivity towards metal ions. Our results indicate that the short form of DPP9 can be isolated from bovine testes and that it behaves as a stable enzyme suitable for further functional and biochemical characterization as well as for inhibitor screening and characterization. 相似文献