首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC‐TR), which crosslinks additional trypsin molecules onto covalently attached trypsin (CA‐TR). EC‐TR showed better stability than CA‐TR in rigorous conditions, such as at high temperatures of 40 and 50°C, in the presence of organic co‐solvents, and at various pH's. For example, the half‐lives of CA‐TR and EC‐TR were 1.42 and 231 h at 40°C, respectively. The improved stability of EC‐TR can be explained by covalent linkages on the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40°C by using both CA‐TR and EC‐TR in digesting a model protein, enolase. EC‐TR showed better performance and stability than CA‐TR by maintaining good performance of enolase digestion under recycled uses for a period of 1 week. In the same condition, CA‐TR showed poor performance from the beginning and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic analysis but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes. Biotechnol. Bioeng. 2010;107: 917–923. © 2010 Wiley Periodicals, Inc.  相似文献   

2.
In this work, a novel and facile route was developed for the immobilization of enzyme on nanosized magnetic particles, and its application to fast protein digestion via a direct MALDI-TOF mass spectrometry analysis was demonstrated. At first, amine-functionalized magnetic particles with high magnetic responsivity and excellent dispersibility were prepared through a facile one-pot strategy. Then, magnetic nanoparticles were functionalized with numerous aldehyde(-CHO) groups by treating the as-synthesized, amine-functionalized magnetic nanoparticles with glutaraldehyde. Finally, immobilization of trypsin onto the aldehyde-functionalized magnetic nanoparticles was achieved through reaction of the aldehyde groups with amine groups of trypsin. The obtained trypsin-immobilized magnetic nanoparticles were conveniently applied for protein digestion. The digestion efficiency was demonstrated with peptide mapping analysis of three model proteins. The process of digestion is very facile due to the easy manipulation of magnetic nanoparticles. Complete protein digestion was achieved in a short time (5 min), without any complicated reduction and alkylation procedures. These results are expected to open up a new possibility for the proteolysis analysis as well as a new application of magnetic nanoparticles. Additionally, it is worth noting that, since the preparation and surface functionality of magnetic nanoparticles is low-cost and reproducible, the preparation method and application approach of the magnetic nanoparticles may find much potential in proteome research.  相似文献   

3.
In this work, polydopamine‐coated magnetic graphene (MG@PDA) nanocomposites were synthesized by a facile method. Trypsin was then directly immobilized on the surface of the nanocomposites through simple PDA chemistry with no need for introducing any other coupling groups. The as‐made MG@PDA nanocomposites inherit not only the large surface area of graphene which makes them capable of immobilizing high amount of trypsin (up to 0.175 mg/mg), but also the good hydrophilicity of PDA which greatly improves their biocompatibility. Moreover, the strong magnetic responsibility makes them easy to be separated from the digested peptide solution when applying a magnetic field. The feasibility of the trypsin‐immobilized MG@PDA (MG@PDA‐trypsin) nanocomposites for protein digestion was investigated and the results indicated their high digestion efficiency in a short digestion time (10 min). In addition, the reusability and stability of the MG@PDA‐trypsin nanocomposites were also tested in our work. To further confirm the efficiency of MG@PDA‐trypsin nanocomposites for proteome analysis, they were applied to digest proteins extracted from skimmed milk, followed by nano RPLC‐ESI‐MS/MS analysis, and a total of 321 proteins were identified, much more than those obtained by 16‐h in‐solution digestion (264 proteins), indicating the great potential of MG@PDA‐trypsin nanocomposites as the supports for high‐throughput proteome study.  相似文献   

4.
Here we present a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI–TOF/TOF)-based label-free relative protein quantification strategy that involves sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) separation of proteins followed by in-gel trypsin digestion. The main problem encountered in gel-based protein quantification is the difficulty in achieving complete and consistent proteolytic digestion. To solve this problem, we developed a high-pressure-assisted in-gel trypsin digestion method that is based on pressure cycling technology (PCT). The PCT approach performed at least as well as the conventional overnight in-gel trypsin digestion approach in parameters such as number of peaks detected, number of peptides identified, and sequence coverage, and the digestion time was reduced to 45 min. The gel/mass spectrometry (MS)-based label-free protein quantification method presented in this work proved the applicability of the signal response factor concept for relative protein quantification previously demonstrated by other groups using the liquid chromatography (LC)/MS platform. By normalizing the average signal intensities of the three most intense peptides of each protein with the average intensities of spiked synthetic catalase tryptic peptides, which we used as an internal standard, we observed spot-to-spot and lane-to-lane coefficients of variation of less than 10 and 20%, respectively. We also demonstrated that the method can be used for determining the relative quantities of proteins comigrating during electrophoretic separation.  相似文献   

5.
Immobilized trypsin (IM) has been recognized as an alternative to free trypsin (FT) for accelerating protein digestion 30 years ago. However, some questions of IM still need to be answered. How does the solid matrix of IM influence its preference for protein cleavage and how well can IM perform for deep bottom‐up proteomics compared to FT? By analyzing Escherichia coli proteome samples digested with amine or carboxyl functionalized magnetic bead–based IM (IM‐N or IM‐C) or FT, it is observed that IM‐N with the nearly neutral solid matrix, IM‐C with the negatively charged solid matrix, and FT have similar cleavage preference considering the microenvironment surrounding the cleavage sites. IM‐N (15 min) and FT (12 h) both approach 9000 protein identifications (IDs) from a mouse brain proteome. Compared to FT, IM‐N has no bias in the digestion of proteins that are involved in various biological processes, are located in different components of cells, have diverse functions, and are expressed in varying abundance. A high‐throughput bottom‐up proteomics workflow comprising IM‐N‐based rapid protein cleavage and fast CZE‐MS/MS enables the completion of protein sample preparation, CZE‐MS/MS analysis, and data analysis in only 3 h, resulting in 1000 protein IDs from the mouse brain proteome.  相似文献   

6.
In the present work, we report a novel on‐target protein cleavage method. The method utilizes ultrasonic energy and allows up to 20 samples to be cleaved in 5 min for protein identification and one sample in 30 s for on‐tissue digestion. The standard proteins were spotted on a conductive glass slide in a volume of 0.5 μL followed by 5 min of ultrasonication after trypsin addition. Controls (5 min, 37°C no ultrasonication) were also assayed. After trypsin addition, digestion of the tissues was enhanced by 30 s of ultrasonication. The samples were analyzed and compared to those obtained by using conventional 3 h heating proteolysis. The low sample volume needed for the digestion and reduction in sample‐handling steps and time are the features that make this method appealing to the many laboratories working with high‐throughput sample treatment.  相似文献   

7.
The susceptibility of proteins in the myelin membrane to proteases was studied. Lyophilized rat brain myelin suspended in water was subjected to controlled proteolytic digestion with pure trypsin (N-tosyl-L-phenylalanine chloromethyl ketone treated, 5 units/mg of myelin), and proteins remaining in the pellet were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under these conditions, large basic protein (LBP) was completely hydrolyzed in 5-10 min, proteolipid proteins remained largely intact until 60 min, whereas Wolfgram protein (WP) was progressively degraded from 10 min onward with the simultaneous appearance of a new protein band with a molecular weight of 35K. A similar pattern was obtained on treatment with chymotrypsin or subtilisin. The 35K protein band was shown to be derived from WP by its immunological cross-reactivity with WP antibodies. Western blot analysis showed that 35K protein is the only major breakdown product of WP under these conditions. Treatment with higher concentrations of trypsin (greater than 20 units/mg of myelin) resulted in the degradation of all myelin proteins. Essentially all the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) activity was observed in the myelin pellet after controlled or drastic digestion with trypsin. It is concluded that the major fragment of WP (35K) is located in the hydrophobic milieu of the bilayer, relatively inaccessible to trypsin, whereas a portion (20K) of the WP is exposed to the cytoplasmic side (major dense line), like LBP, and that peptide fragments (less than 14K) that remained in the myelin membrane lipid bilayer after trypsin digestion could exhibit CNP activity.  相似文献   

8.
In this study, a novel microwave-assisted protein digestion method was developed using trypsin-immobilized magnetic nanoparticles (TIMNs). The magnetic nanoparticles worked as not only substrate for enzyme immobilization, but also excellent microwave irradiation absorber and, thus, improved the efficiency of microwave-assisted digestion greatly. Three standard proteins, bovine serum albumin (BSA), myoglobin, and cytochrome c, were used to optimize the conditions of this novel digestion method. With the optimized conditions, peptide fragments produced in very short time (only 15 s) could be identified successfully by MALDI-TOF-MS. When it was compared to the conventional in-solution digestion (12 h), equivalent or better digestion efficiency was observed. Even when protein quantity was as low as micrograms, this novel digestion method still could digest proteins successfully, while the same samples by conventional in-solution digestion failed. Moreover, with an external magnetic field, the enzyme could be removed easily and reused. It was verified that, after 4 replicate runs, the TIMNs still kept high activity. To further confirm the efficiency of this rapid digestion method for proteome analysis, it was applied to the protein extract of rat liver. Without any preparation and prefractionation processing, the entire proteome digested by TIMNs in 15 s went through LC-ESI-MS/MS direct analysis. The whole shotgun proteomic experiment was finished in only 1 h with the identification of 313 proteins ( p < 0.01). This new application of TIMNs in microwave-assisted protein digestion really opens a route for large-scale proteomic analysis.  相似文献   

9.
Human norovirus is a leading cause of acute gastroenteritis worldwide in a plethora of residential and commercial settings, including restaurants, schools, and hospitals. Methods for easily detecting the virus and for treating and preventing infection are critical to stopping norovirus outbreaks, and inactivation via nanoparticles (NPs) is a more universal and attractive alternative to other physical and chemical approaches. Using norovirus GI.1 (Norwalk) virus-like particles (VLPs) as a model viral system, this study characterized the antiviral activity of Au/CuS core/shell nanoparticles (NPs) against GI.1 VLPs for the rapid inactivation of HuNoV. Inactivation of VLPs (GI.1) by Au/CuS NPs evaluated using an absorbance-based ELISA indicated that treatment with 0.083 μM NPs for 10 min inactivated ~50% VLPs in a 0.37 μg/ml VLP solution and 0.83 μM NPs for 10 min completely inactivated the VLPs. Increasing nanoparticle concentration and/or VLP-NP contact time significantly increased the virucidal efficacy of Au/CuS NPs. Changes to the VLP particle morphology, size, and capsid protein were characterized using dynamic light scattering, transmission electron microscopy, and Western blot analysis. The strategy reported here provides the first reported proof-of-concept Au/CuS NPs-based virucide for rapidly inactivating human norovirus.  相似文献   

10.
Three different lines of evidence were obtained to show that trypsin modifies the actin-myosin interaction: (I) At trypsin to actomyosin or myosin ratios between 1 to 300 and 1 to 500, 30 min of trypsin treatment causes an 8-fold increase in the Ca2+-modified ITPase activity of actomyosin but has no effect on the Ca2+-modified ITPase activity of myosin alone. At these same trypsin to actomyosin ratios, the Mg2+ + Ca2+-modified ATPase activity increases by 10–30% during the first 1–2 min of trypsin digestion, and then decreases rapidly to less than 20% of its original activity after 60 min of digestion. Trypsin has no effect on the Mg2+ + Ca2+-modified ATPase activity of pure myosin. (2) The rate of turbidity response of reconstituted actomyosin suspensions is first increased and then decreased by trypsin treatment. At trypsin to actomyosin ratios of 1 to 3000, rate of turbidity response is maximal after 5 min of trypsin digestion and then decreases; after 60 min, the turbidity response is much slower than the response of the control actomyosin. (3) Supercontracted sarcomeres, shortened to less than 50% of their initial length, are lengthened to 70% of their initial length by 4 min of trypsin treatment. Myosin B from such lengthened sarcomeres has less than 35% of its myosin converted to light meromyosin and heavy meromyosin.

These results show that trypsin modifies the actin-myosin interaction in at least two ways: (1) a very rapid initial modification that increases the Mg2+ + Ca2+-modified ATPase activity and the rate of turbidity increase, and (2) a slower modification that decreases the Mg2+ + Ca2+-modified ATPase activity and rate of turbidity response, and that lengthens contracted sarcomeres. Tryptic modification is not due to cleavage of myosin to light and heavy meromyosin. Since tryptic modification occurs more rapidly than conversion of myosin to light and heavy meromyosin, all heavy meromyosin preparations will be modified.  相似文献   


11.
In proteome research, rapid and effective proteolysis and enrichment strategies are essential for successful protein identification. Functionalized magnetic microspheres of micro- and nano-meter size are gaining increasing attention due to their easy manipulation and recovery, great specific surface areas and high surface activity. The introduction of magnetic nanoparticles into the field of proteomics study has accelerated the development of digestion and enrichment methods. In this article, we mainly focus on recent developments of using different functionalized magnetic nanoparticles for rapid digestion and preconcentration of low-abundance peptides/proteins, including those containing post-translational modifications, such as phosphorylation and glycosylation, prior to mass spectrometric analysis.  相似文献   

12.
DigesTip is a new device for in-solution protein digestion, based on a patent pending technology, able to immobilize enzymes (trypsin, in this case) on a solid surface, keeping their activity preserved. DigesTip is a standard pipette tip, usable both by human and by robots. Its main performances are: very short digestion time (1 min) and usability with low protein sample concentrations (5 microg/mL). DigesTip obtains a clear signal in MS measurements and its usage rules out several preparative steps.  相似文献   

13.
A stable and robust trypsin‐based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300‐fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC‐MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in “real‐world” proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.  相似文献   

14.
Trypsin digestion can induce artificial modifications such as asparagine deamidation and N-terminal glutamine cyclization on proteins due to the temperature and the alkaline pH buffers used during digestion. The amount of these artificial modifications is directly proportional to the incubation time of protein samples in the reduction/alkylation buffer and, more important, in the digestion buffer where the peptides are completely solvent exposed. To minimize these artificial modifications, we focused on minimizing the trypsin digestion time by maximizing trypsin activity. Trypsin activity was optimized by the complete removal of guanidine, which is a known trypsin inhibitor, from the digestion buffer. As a result, near complete trypsin digestion was achieved on reduced and alkylated immunoglobulin gamma molecules in 30 min. The protein tryptic fragments and their modification products were analyzed and quantified by reversed-phase liquid chromatography/tandem mass spectrometry using an in-line LTQ Orbitrap mass spectrometer. The reduction and alkylation reaction time was also minimized by monitoring the completeness of the reaction using a high-resolution time-of-flight mass spectrometer. Using this 30-min in-solution trypsin digestion method, little protocol-induced deamidation or N-terminal glutamine cyclization product was observed and cleaner tryptic maps were obtained due to less trypsin self-digestion and fewer nonspecific cleavages. The throughput of trypsin digestion was also improved significantly compared with conventional trypsin digestion methods.  相似文献   

15.
Various nanoparticles, such as silver nanoparticles (AgNPs) and titanium nanoparticles (TiO2NPs) are increasingly used in industrial processes. Because they are released into the environment, research into their influence on the biosphere is necessary. Among its other effects, dietary TiO2NPs promotes silk protein synthesis in silkworms, which prompted our hypothesis that TiO2NPs influence protein kinase B (Akt)/Target of rapamycin (Tor) signaling pathway (Akt/Tor) signaling in their silk glands. The Akt/Tor signaling pathway is a principle connector integrating cellular reactions to growth factors, metabolites, nutrients, protein synthesis, and stress. We tested our hypothesis by determining the influence of dietary TiO2NPs (for 72 h) and, separately, of two Akt/Tor pathway inhibitors (LY294002 and rapamycin) on expression of Akt/Tor signaling pathway genes and proteins in the silk glands. TiO2NPs treatments led to increased accumulation of mRNAs for Akt, Tor1 and Tor2 by 1.6‐, 12.1‐, and 4.8‐fold. Dietary inhibitors led to 2.6‐ to 4‐fold increases in mRNAs encoding Akt and substantial decreases in mRNAs encoding Tor1 and Tor2. Western blot analysis showed that dietary TiO2NPs increased the phosphorylation of Akt and its downstream proteins. LY294002 treatments led to inhibition of Akt phosphorylation and its downstream proteins and rapamycin treatments similarly inhibited the phosphorylation of Tor‐linked downstream proteins. These findings support our hypothesis that TiO2NPs influence Akt/Tor signaling in silk glands. The significance of this work is identification of specific sites of TiO2NPs actions.  相似文献   

16.
Improving the lithium (Li) storage properties of silicon (Si)‐based anode materials is of great significance for the realization of advanced Li‐ion batteries. The major challenge is to make Si‐based anode materials maintain electronic conduction and structural integrity during cycling. Novel carbon‐coated Si nanoparticles (NPs)/reduced graphene oxides (rGO) composites are synthesized through simple solution mixing and layer‐by‐layer assembly between polydopamine‐coated Si NPs and graphene oxide nanosheets by filtration, followed by a thermal reduction. The anodic properties of this composite demonstrate the potency of the novel hybrid design based on two dimensional materials for extremely reversible energy conversion and storage. A high capacity and an extremely stable cycle life are simultaneously realized with carbon‐coated Si/rGO composite, which has a sandwich structure. The unprecedented electrochemical performance of this composite can be ascribed to the synergistic effect of polydopamine and rGO. The polydopamine layer forms strong hydrogen bonding with rGO through chemical cross‐linking, thus firmly anchoring Si NPs on rGO sheets to prevent the aggregation of Si NPs and their electronic contact loss. Finally, its structural feature with stacked rGO clipping carbon‐coated Si NPs inside it enables to keep the overall electrode highly conductive and mechanically robust, thus maintaining its initial capacity even with extended cycling.  相似文献   

17.
Li Y  Yan B  Deng C  Tang J  Liu J  Zhang X 《Proteomics》2007,7(20):3661-3671
In this study, a novel method of on-plate digestion using trypsin-immobilized magnetic nanospheres was developed followed by MALDI-TOF-MS for rapid and effective analysis and identification of proteins. We utilized a facile one-pot method for the direct preparation of amine-functionalized magnetic nanospheres with highly magnetic properties and the amino groups on the outer surface. Through the reaction of the aldehyde groups with amine groups, trypsin was simply and stably immobilized onto the magnetic nanospheres. The obtained trypsin-linked magnetic nanospheres were then applied for on-plate digestion of sample proteins (myoglobin and Cytochrome c). Moreover, after digestion, the trypsin-linked nanospheres could be easily removed from the plate due to their magnetic property, which would avoid causing contamination on the ion source chamber in MS. The effects of the temperature and incubation time on the digestion efficiency were characterized. Within only 5 min, proteins could be efficiently digested with the peptide sequence coverage higher than or equal to that of the traditional in-solution digestion for 12 h. Furthermore, RPLC fractions of rat liver extract were also successfully processed using this novel method. These results suggested that our improved on-plate digestion protocol for MALDI-MS may find further application in automated analysis of large sets of proteins.  相似文献   

18.
The Ca2+ pumps associated with human platelet plasma and intracellular membranes have been further characterized by their sensitivity to trypsin. (a) Tryptic degradation of the Ca2+-ATPases has been followed by immunoblotting. It resulted in fragmentation into peptides of 80, 55, 35, and 24 kDa for both enzymes. Subcomplete hydrolysis obtained with a ratio of trypsin/membrane protein of 0.05-0.1 for the two Ca2+ pumps resulted in the total disappearance of the 100-, 80-, and 35-kDa fragments. However, maximum degradation was reached within 1 min for the intracellular enzyme but needed 5 min of incubation for the plasma membrane enzyme. (b) This effect of trypsin has been correlated with its effect on both the Ca2+-ATPase activities. The plasma membrane enzyme showed a maximum inhibition of 50-60% which was obtained using a trypsin/protein ratio of 0.1 and 5 min of incubation. A much higher trypsin sensitivity was observed for the intracellular enzyme because the maximum inhibition reached 80% after only 1 min of incubation. (c) Finally, the two Ca2+ transport systems studied showed different trypsin reactivities; the Ca2+ uptake by the plasma membrane vesicles was inhibited by 20-25%, and this maximum inhibition was observed after 5 min of incubation with trypsin. In contrast, the Ca2+ transport associated with the intracellular membrane vesicles was difficult to detect after trypsin treatment. Taken together, the results show that the two Ca2+ pumps can be distinguished by their trypsin sensitivity.  相似文献   

19.
Plasma is generated by ionizing gas molecules. Helium (He)‐based cold atmospheric plasma (CAP) was generated using a high‐voltage power supply with low‐frequency excitation (60 Hz at 7 kV) and He flow at 2 l/min. Platinum nanoparticles (Pt‐NPs) are potent antioxidants due to their unique ability to scavenge superoxides and peroxides. These features make them useful for the protection against oxidative stress‐associated pathologies. Here, the effects of Pt‐NPs on He‐CAP‐induced apoptosis and the underlying mechanism were examined in human lymphoma U937 cells. Apoptosis was measured after cells were exposed to He‐CAP in the presence or absence of Pt‐NPs. The effects of combined treatment were determined by observing the changes in intracellular reactive oxygen species (ROS) and both mitochondrial and Fas dependent pathway. The results indicate that Pt‐NPs substantially scavenge He‐CAP‐induced superoxides and peroxides and inhibit all the pathways involved in apoptosis execution. This might be because of the SOD/catalase mimetic effects of Pt‐NPs. These results showed that the Pt‐NPs can induce He‐CAP desensitization in human lymphoma U937 cells.  相似文献   

20.
There is a relationship between the normal progress of digestion and the retention or elimination of the proteins ingested with the meal by Aedes aegyti females. The addition of soybean trypsin inhibitor (STI) to a protein meal prevented digestion and resulted in a rapid elimination of the undigested proteins. The addition of a mix of free amino acids to a protein meal together with STI resulted in a significant increase in the retention of the undigested proteins during the first 10-15 hrs after feeding. The effect of the free amino acids on the retention of the proteins was concentration-dependent between 250 microg/ml and 5 mg/ml. Free amino acids were also important for the retention of non-protein meals. When females were fed a meal containing FITC-dextran (20 kD), most of this compound was eliminated into the feces by 10 hrs; the addition of free amino acid resulted in a significant increase in the retention of the FITC-dextran by the midgut during the first 15 hrs after feeding. The presence of free amino acids in the midgut lumen seems to be an important signal used by the mosquito to regulate the retention of the meal.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号