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1.
MALDI profiling and imaging mass spectrometry (IMS) are novel techniques for direct analysis of peptides and small proteins in biological tissues. In this work we applied them to the study of Anopheles gambiae antennae, with the aim of analysing expression of soluble proteins involved in olfaction perireceptor events. MALDI spectra obtained by direct profiling on single antennae and by the analysis of extracts, showed similar profiles, although spectra obtained through profiling had a richer ion population and higher signal to noise ratio. Male and female antennae showed distinct protein profiles. MALDI imaging experiments were also performed and differences were observed in the localization of some proteins. Two proteins were identified through high resolution measurement and top-down MS/MS experiments. A 8 kDa protein only present in the male antennae matched with an unannotated sequence of the An. gambiae genome, while the presence of odorant binding protein 9 (OBP-9) was confirmed through experiments of 2-DE, followed by MS and MS/MS analysis of digested spots. This work shows that MALDI MS profiling is a technique suitable for the analysis of proteins of small and medium MW in insect appendices, and allows obtaining data for several specimens which can be investigated for differences between groups. Proteins of interest can be identified through other complementary MS approaches.  相似文献   

2.
Previous studies have shown that increases in surface-peptide binding affinity result in decreases in peptide matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) ion signals. The present work demonstrates that, with appropriate corrections for peptide ionization efficiency under MALDI conditions, relative surface-peptide binding affinities can be assayed using the MALDI MS methodology. Peptides with a range of pI values are allowed to interact with amine-modified and carboxylic acid-modified polymer surfaces (produced by pulsed radio-frequency plasma polymerization of allyl amine and vinyl acetic acid) in buffered solutions of neutral pH. Because of the net positive and negative charges associated with the peptides and surfaces in solution, both electrostatic and hydrophilic interactions play a role in the surface-peptide interaction. Consistent with expectations, the peptide MALDI ion signals for peptides with net negative charges in solution are smaller than those for peptides with net positive charges in solution when the peptides are allowed to interact with positively charged surfaces. A reversal of the relative peptide MALDI ion signal intensities is observed when the same peptides are allowed to interact with negatively charged surfaces. Cumulatively, the results demonstrate that even modest changes in surface-peptide interactions can be comparatively probed by MALDI mass spectrometry.  相似文献   

3.
On-target affinity capture, enrichment and purification of biomolecules improve detection of specific analytes from complex biological samples in matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. In this paper, we report a simple method for preparation of a self-assembled nitrilotriacetic acid (NTA) monolayer on gold surface which can be used as a MALDI-TOF-MS sample target specifically for recombinant oligohistidine-tagged proteins/peptides and phosphorylated peptides. The NTA functional groups are immobilized to the gold surface via the linkage of 1,8-octanedithiol which forms a self-assembled monolayer on gold. Characterization by X-ray photoelectron spectroscopy and MALDI analysis of the modified surface are described. The chemically modified surface shows strong affinity toward the analytes of interest, which allows effective removal of the common interferences, e.g. salts and detergents, and therefore leads to improved signal/noise ratio and detection limit. The use of the modified surface simplifies the sample preparation for MALDI analysis of these targeted analytes.  相似文献   

4.
Sputtered silicon nitride optical waveguide surfaces were silanized and modified with a hetero-bifunctional crosslinker to facilitate thiol-reactive immobilization of contact-printed DNA probe oligonucleotides, streptavidin and murine anti-human interleukin-1 beta capture agents in microarray formats. X-ray photoelectron spectroscopy (XPS) was used to characterize each reaction sequence on the native silicon oxynitride surface. Thiol-terminated DNA probe oligonucleotides exhibited substantially higher surface printing immobilization and target hybridization efficiencies than non-thiolated DNA probe oligonucleotides: strong fluorescence signals from target DNA hybridization supported successful DNA oligonucleotide probe microarray fabrication and specific capture bioactivity. Analogously printed arrays of thiolated streptavidin and non-thiolated streptavidin did not exhibit noticeable differences in either surface immobilization or analyte capture assay signals. Non-thiolated anti-human interleukin-1 beta printed on modified silicon nitride surfaces reactive to thiol chemistry exhibited comparable performance for capturing human interleukin-1 beta analyte to commercial amine-reactive microarraying polymer surfaces in sandwich immunoassays, indicating substantial non-specific antibody-surface capture responsible for analyte capture signal.  相似文献   

5.
Tryptic digestion of proteins continues to be a workhorse of proteomics. Traditional tryptic digestion requires several hours to generate an adequate protein digest. A number of enhanced accelerated digestion protocols have been developed in recent years. Nonetheless, a need still exists for new digestion strategies that meet the demands of proteomics for high-throughput and rapid detection and identification of proteins. We performed an evaluation of direct tryptic digestion of proteins on a MALDI target plate and the potential for integrating RP HPLC separation of protein with on-target tryptic digestion in order to achieve a rapid and effective identification of proteins in complex biological samples. To this end, we used a Tempo HPLC/MALDI target plate deposition hybrid instrument (ABI). The technique was evaluated using a number of soluble and membrane proteins and an MRC5 cell lysate. We demonstrated that direct deposition of proteins on a MALDI target plate after reverse-phase HPLC separation and subsequent tryptic digestion of the proteins on the target followed by MALDI TOF/TOF analysis provided substantial data (intact protein mass, peptide mass and peptide fragment mass) that allowed a rapid and unambiguous identification of proteins. The rapid protein separation and direct deposition of fractions on a MALDI target plate provided by the RP HPLC combined with off-line interfacing with the MALDI MS is a unique platform for rapid protein identification with improved sequence coverage. This simple and robust approach significantly reduces the sample handling and potential loss in large-scale proteomics experiments. This approach allows combination of peptide mass fingerprinting (PMF), MS/MS peptide fragment fingerprinting (PPF) and whole protein MS for both protein identification and structural analysis of proteins.  相似文献   

6.
After their translation and folding in the cytoplasm, proteins may be imported into an organelle, associate with a membrane, or rather become part of large, highly localised cytoplasmic structures such as the cytoskeleton. The localisation of a protein is governed by the strength of binding to its immediate target, such as an import receptor for an organelle or a major component of the cytoskeleton, e.g. actin. We have experimentally provided a set of actin-binding proteins with competing targeting information and expressed them at various concentrations to analyse the strength of the signal that governs their subcellular localisation. Our microscopic observations indicate that organellar sorting signals override the targeting preference of most cytoskeletal proteins. Among these signals, the nuclear localisation signal of SV40 is strongest, followed by the oligomerised PHB domain that targets vacuolin to the endosomal surface, and finally the tripeptide SKL mediating transport into the peroxisome. The actin-associated protein coronin, however, can only be misled by the nuclear localisation signal. Interestingly, the targeting behaviour of this model set of hybrid proteins in living Dictyostelium amoebae correlates surprisingly well with the affinities of their constituent signals derived from in vitro experiments conducted in various other organisms. Accordingly, this approach allows estimating the in vivo affinity of a protein to its target even if the latter is not known, as in the case of vacuolin.  相似文献   

7.
Recent advances in MALDI MS/MS instrumentation allow a high degree of automation in the efficient detection of peptide fragment ions that can be used for protein identification. However, the performance of the technique is dependent on the MALDI sample preparation. We present a simple and robust two-layer sample preparation method tailored for sensitive and reproducible generation of MALDI MS/MS data. This method produces a strong and uniform crystal layer which allows acquisition of high quality MS/MS spectra over the entire sample surface area. Furthermore, due to its crystal strength, the matrix/sample layer can be washed extensively on target, enabling direct analysis of samples containing impurities, such as salts and surfactants. This method is demonstrated to be very useful in routine analysis of in-gel tryptic digests of silver-stained protein gel spots, without the need of desalting steps or hunting for "hot" spots. As an example, seven threonine-phosphorylated proteins involved in signal transduction in response to growth factor stimulation within the lipid raft fractions of the IMR5 neuroblastoma cells have been identified using differential gel display, in-gel digestion and MALDI MS/MS with the new two-layer sample preparation method. Some of these proteins have the functions of maintaining raft structure or cell signaling.  相似文献   

8.
A method is presented for the recovery and subsequent guanidination of tryptic peptides from samples previously spotted on a matrix-assisted laser desorption/ionization (MALDI) target. The procedure is shown to have applicability to both in-solution and in-gel digests, yielding improved confidence in protein identification and sequence coverage in all instances. Recovery from the plate is essentially quantitative, with no residual analyte observed on the target spot. The technique is rapid, simple, and has extended applicability to other processing steps, including (but not limited to) derivatization for specific peptide studies or enzymatic treatment for subsequent profiling of posttranslational modifications. This method circumvents the failure of an initial analysis to generate suitable information and is particularly relevant for the analysis of precious samples.  相似文献   

9.
A mathematical model for metal affinity protein partitioning   总被引:2,自引:0,他引:2  
A mathematical model of metal affinity partitioning has been derived and used to describe protein partitioning in Cu (II)PEG/dextran systems. A working model has been extended to account for inhibition, which for metal affinity extraction is the inhibition of protein-metal binding by hydrogen ion. PEG/dextran partitioning experiments were performed on four proteins, tuna heart cytochrome c, Candida krusei cytochrome c, horse myoglobin, and sperm whale myoglobin. The partition coefficients for these proteins are increased by the addition of Cu (II)PEG-IDA, due to the affinity between the chelated copper atom and metal-coordinating histidine residues on the protein surface. The results of experiments to determine the effects of the number of binding sites on the protein, the copper concentration, and pH on partitioning are all well-described by the mathematical model. The pK(a) value of the metal binding site was determined to be 6.5, which is in the range of pK(a) values commonly observed for surface histidines. The average association constant for the binding of Cu (II)PEG-IDA to accessible histidines was found to be 4.5 x 10(3). This value is comparable to stability constants measured by conventional potentiometry techniques for analogous small complexes.  相似文献   

10.
Nedelkov D  Nelson RW 《Proteomics》2001,1(11):1441-1446
Biomolecular interaction analysis mass spectrometry (BIA-MS) is a multiplexed bioanalytical approach used in analysis of proteins from complex biological mixtures. It utilizes surface-immobilized ligands for protein affinity retrieval, surface plasmon resonance for monitoring the ligand-protein interaction and matrix-assisted laser desorption/ionization-time of flight mass spectrometry for revealing the masses of the biomolecules retrieved by the ligand. In order to explore the utility of BIA-MS in delineation of multiprotein complexes, an in vivo assembled protein complex comprised of retinol binding protein (RBP) and transthyretin (TTR) was investigated. Antibodies to RBP and TTR were utilized as ligands in the analysis of the protein complex present in human plasma. The RBP-TTR complex was retrieved by the anti-RBP antibody as indicated by the presence of both RBP and TTR signals in the mass spectra. RBP signals were not observed in the mass spectra of the material retained on the anti-TTR derivatized surface. In addition, the mass-specific detection in BIA-MS allowed detection of RBP and TTR analyte variants.  相似文献   

11.
Identification of ubiquitinated proteins in Arabidopsis   总被引:1,自引:0,他引:1  
Ubiquitin (Ub) is a small peptide that is covalently attached to proteins in a posttranslational reaction. Ubiquitination is a precise regulatory system that is present in all eukaryotic organisms and regulates the stability, the activity, the localization and the transport of proteins. Ubiquitination involves different enzymatic activities, in which the E3 ligases catalyze the last step recruiting of the target for labelling with ubiquitin. Genomic analyses have shown that the ubiquitin-proteasome system involves a large number of proteins in plants, as approximately 5% of the total protein belongs to this pathway. In contrast to the high number of E3 ligases of ubiquitin identified, very few proteins regulated by ubiquitination have been described. To solve this, we have undertaken a new proteomic approach aimed to identify proteins modified with ubiquitin. This is based on affinity purification and identification for ubiquitinated proteins using the ubiquitin binding domain (UBA) polypeptide of the P62 protein attached to agarose beads. This P62-agarose matrix is capable of specifically binding ubiquitinated proteins. These bound proteins were digested with trypsin and the peptides separated by HPLC chromatography, spotted directly onto a MALDI target and analyzed by MALDI-TOF/TOF off-line coupled LC/MALDI-MS/MS. A total of 200 putative ubiquitinated proteins were identified. From these we found that several of the putative targets were already described in plants, as well as in other organisms, as ubiquitinated proteins. In addition, we have found that some of these proteins were indeed modified with ubiquitin in vivo. Taken together, we have shown that this approach is useful for identifying ubiquitinated protein in plants.  相似文献   

12.
Spectroscopic methods have been employed in order to understand the molecular basis of the decrease in enzymatic activity of the antiinflammatory enzyme copper-zinc superoxide dismutase (SOD) following the covalent binding of polyethyleneglycol (PEG) chains to the protein amino-groups. The PEG modification is a general method recently proposed to improve the therapeutic index of enzymes. 1H NMR spectra on the cobalt substituted PEG-modified SOD, Cu2Co2-PEG-SOD, have been recorded. The signals are quite broad with respect to the unmodified enzyme. This has been interpreted on the basis of the effect of molecular weight on the linewidth. The analysis has shown that the histidine hydrogens involved in metal binding at the enzyme active site are the same in both native and PEG-modified SOD. Similarly, circular dichroism and absorption spectra indicate that the overall conformation of the metal clusters is not perturbed upon modification. On the other hand, azide titration shows that the affinity constant of N-3 for SOD is largely reduced upon PEG modification (K = 154 M-1 and 75 M-1 for the native and modified SOD, respectively). These results indicate that the decrease in enzymatic activity upon surface modification with PEG is not caused by a perturbation of the active site geometry, but to a decrease in the channeling of the O2- ion towards the enzyme active site.  相似文献   

13.
Integrating surface plasmon resonance analysis with mass spectrometry allows detection and characterization of molecular interactions to be complemented with identification of interaction partners. We have developed a procedure for Biacore 3000 that automatically performs all steps from ligand fishing and recovery to sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry including on-target digestion. In the model system used in this study a signal transduction protein, calmodulin, was selectively captured from brain extract by one of its interaction partners immobilized on a sensor chip. The bound material was eluted, deposited directly onto a MALDI target, and analyzed by mass spectrometry both as an intact protein and after on-target tryptic digestion. The procedure with direct deposition of recovered material on the MALDI target reduces sample losses and, in combination with automatic sample processing, increases the throughput of surface plasmon resonance mass spectrometry analysis.  相似文献   

14.
Surface plasmon resonance (SPR) can detect molecules bound to a surface by subtle changes in the SPR angle. By immobilizing probes onto the surface and passing analyte solution through the surface, changes in SPR angle indicate the binding between analyte and probes. Detection of analyte from solution can be achieved easily. By using rolling circle amplification (RCA) and nanogold-modified tags, the signals of analyte binding are greatly amplified, and the sensitivity of this technique is significantly improved. Furthermore, this technique has potentials for ultra-sensitive detection and microarray analysis. In this paper, this detection technique is introduced and shown to have great amplification capability. Using 5 nm nanogold with 30 min of RCA development time, this proposed protein detection technique shows over 60 times amplification of the original signal.  相似文献   

15.
Being able to rapidly and sensitively detect specific enzymatic products is important when screening biological samples for enzymatic activity. We present a simple method for assaying protease activity in the presence of protease inhibitors (PIs) by measuring tryptic peptide accumulation on copolymer pMALDI target chips using a dual fluorescence/MALDI‐TOF‐MS read‐out. The small platform of the chip accommodates microliter amounts of sample and allows for rapid protein digestion. Fluorescamine labeling of tryptic peptides is used to indicate the proteolytic activity and is shown to be an affordable, simple process, yielding a strong fluorescence signal with a low background. Subsequent MALDI‐TOF‐MS analysis, performed in the same sample well, or in a parallel well without adding fluorescamine, detects the specific tryptic peptides and provides confidence in the assay. The dual read‐out method was applied to screen the inhibition activity of plant PIs, components of plant defense against herbivores and pathogens. Extracts of PIs from Solanum nigrum and trypsin were applied together to a pMALDI chip on which a suitable substrate was adsorbed. The fluorescence and MALDI‐TOF‐MS signal decrease were associated with the inhibitory effect of the PIs on trypsin. The developed platform can be modified to screen novel protease inhibitors, namely, those potentially useful for treating or preventing infection by viruses, including HIV and hepatitis C.  相似文献   

16.
Mark L. Stolowitz 《Proteomics》2012,12(23-24):3438-3450
Over the course of the last decade, a number of investigators have come to appreciate that the surface of a MALDI target, after suitable modification, can be used for selective enrichment of peptides and proteins. More recently, surface‐modified nanoparticles (NPs) that readily co‐crystallize in MALDI matrix, are not ionized by laser desorption/ionization, and do not interfere with MS have attracted interest as alternatives to surface‐modified targets for selective enrichment of peptides and proteins. Surface‐modified targets and NPs facilitate parallel processing of samples, and when used in conjunction with MALDI mass spectrometers with kHz lasers enable development of high‐throughput proteomics platforms. Targets and NPs for reversed phase and ion exchange retention, selective enrichment of glycopeptides, selective enrichment of phosphopeptides, and immunoaffinity MS are described in conjunction with details regarding their preparation and utility. Commercial availability of the reagents and substrates required to prepare surface‐modified targets and NPs is also discussed.  相似文献   

17.
Proteolytic digestion of proteins bound to immobilized antibodies, combined with matrix assisted laser desorption (MALDI) mass spectrometric identification of the affinity-bound peptides, can be a powerful technique for epitope determination. Binding of the protein to the antibody is done while the protein is in its native, folded state. A purified protein is not required for this procedure, because only proteins containing the antigenic determinant will bind to the antibody in the initial step. The method makes use of the resistance of the antibody to enzymatic digestion. Enzymatic cleavage products of the antigenic protein not containing the epitope are washed off the beads, leaving the epitope-containing fragments affinity bound to the immobilized antibody. Dissociation of the antigen-antibody complex prior to mass spectrometric analysis is unnecessary because the affinity-bound peptides are released by the MALDI matrix crystallization process, although the antibody remains covalently attached to the sepharose beads. This epitope-mapping protocol has been used in the determination of both continuous and discontinuous epitopes on both glycosylated and unglycosylated proteins.  相似文献   

18.
Protein abundance changes during disease or experimental perturbation are increasingly analyzed by label-free LC/MS approaches. Here we demonstrate the use of LC/MALDI MS for label-free detection of protein expression differences using Escherichia coli cultures grown on arabinose, fructose or glucose as a carbon source. The advantages of MALDI, such as detection of only singly charged ions, and MALDI plate archiving to facilitate retrospective MS/MS data collection are illustrated. MALDI spectra from RP chromatography of tryptic digests of the E. coli lysates were aligned and quantitated using the Rosetta Elucidator system. Approximately 5000 peptide signals were detected in all LC/MALDI runs spanning over 3 orders of magnitude of signal intensity. The average coefficients of variation for all signals across the entire intensity range in all technical replicates were found to be <25%. Pearson correlation coefficients from 0.93 to 0.98 for pairwise comparisons illustrate high replicate reproducibility. Expression differences determined by Analysis of Variance highlighted over 500 isotope clusters ( p < 0.01), which represented candidates for targeted peptide identification using MS/MS. Biologically interpretable protein identifications that could be derived underpin the general utility of this label-free LC/MALDI strategy.  相似文献   

19.
The development of new high throughput methods based on different materials with chemical modifications for protein profiling of complex mixtures leads towards biomarkers; used particularly for early diagnosis of a disease. In this work, diamond-like carbon (DLC) is developed and optimized for serum protein profiling by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). This study is carried out in connection with a material-based approach, termed as material-enhanced laser desorption ionization mass spectrometry. DLC is selected as carrier surface which provides large surface to volume ratio and offers high sensitivity. DLC has a dual role of working as MALDI target while acting as an interface for protein profiling by specifically binding peptides and proteins out of serum samples. Serum constituents are bound through immobilized metal ion affinity chromatography (IMAC) functionality, created through glycidyl methacrylate polymerization under ultraviolet light followed by further derivatization with iminodiacetic acid and copper ion loading. Scanning electron microscopy highlights the morphological characteristics of DLC surface. It could be demonstrated that IMAC functionalized DLC coatings represent a powerful material in trapping biomolecules for their further analysis by MALDI-MS resulting in improved sensitivity, specificity and capacity in comparison to other protein-profiling methods.  相似文献   

20.
A novel linear ion trap (LIT) mass spectrometer with dual matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) ionization sources has been built in the MALDI-LIT-ESI configuration. The design features two independent ion source/ion optical channels connected to opposite ends of a single mass analyzer. The instrument consists of a commercial MALDI-LIT instrument modified by the addition of a home-built vacuum manifold, ion optical system, control electronics, and programming necessary to couple an atmospheric pressure interface to the commercial instrument. In addition to the added ESI functionality, the capabilities of the system also include simultaneous dual-channel ion introduction and analysis and high-duty cycle electronic switching (<1 s) between ion channels. Analytical and ion chemical applications of the dual-source system are explored. One analytical application is the enhanced protein sequence coverage achieved when using both ESI and MALDI to examine a tryptic digest of a six-protein mixture. The differences in the efficiency with which peptides in a mixture are ionized by the two methods give improved sequence coverage when both are applied. Other analytical applications include the use of the ions from one source as intensity or mass standards for the analyte ions from the other. An ion chemistry application involves the use of energy-resolved tandem mass spectrometry (MS/MS) to seek evidence for the generation of isomeric ions from a particular compound using the two ionization methods. A high level of agreement was achieved between the MS/MS spectra recorded under a variety of conditions after ESI and MALDI ionization; this provides evidence of the reproducibility and internal consistency of data from the dual source instrument. However, each of the peptides examined generated identical populations of structures in the two ionization methods under our conditions which are interpreted as involving slow cooling into the most stable minimum on the potential energy surface.  相似文献   

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