A fiber optic particle plasmon resonance (FOPPR) immunosensor is developed for label-free detection of orchid viruses that use gold nanorods (AuNRs) as the sensing material. The AuNRs are employed to ...create a near-infrared sensing window to solve the color interference problem of sample matrix for direct sensing of target analyte. This work cannot be achieved using gold nanospheres (AuNSs) because the signal of sample color absorption largely overlaps the signal of molecular recognition events in the visible spectrum, making the signal interpretation much more difficult. The AuNRs are immobilized on the unclad fiber core surface, and functionalized by antibodies which can specifically recognize the corresponding Cymbidium mosaic virus (CymMV) or Odontoglossum ringspot virus (ORSV) for rapid viral infection diagnosis. The refractive index resolution of the AuNR-FOPPR sensor is estimated to be 8×10−6 RIU. The limits of detection (LODs) for CymMV and ORSV in leaf saps are 48 and 42pg/mL, respectively, which are better than the LODs of 1200pg/mL for both viruses obtained by enzyme-linked immunosorbent assay (ELISA). Exploiting the AuNR-FOPPR sensing strategy not only solves the color interference problem encountered by using AuNSs, but provides faster analysis, better reproducibility, and lower detection limit than ELISA. The sensor can distinguish between healthy and infected orchids in 10min, and can further provide the quantitative analysis of infection level. It is potentially applicable to the quality control of orchid cultivation industry, but not limited to this, especially for creating special spectral sensing window for particular samples.
•A fiber optic particle plasmon resonance immunosensor based on gold nanorods is developed for label-free detection of orchid viruses.•Employing gold nanorods as the sensing material solves the color interference problem encountered by using gold nanospheres.•Faster analysis, better reproducibility, and lower detection limit than ELISA are provided.
Shrimp allergy is a critical public health concern worldwide, and is also the most frequent cause of food allergy in Taiwan. Tropomyosin is recognized as the major shrimp allergen. However, ...information of tropomyosin content in Taiwanese shrimp is unknown. Therefore, it is an urgent need to develop a reliable analytical approach to detect tropomyosin. An absolute quantification method of proteins (AQUA) method was developed to detect tropomyosin in seven Taiwanese shrimps in this study. Both signature peptide, ALSNAEGEVAALNR, and its isotope-labeled peptide were used as standards and internal standards. The determination coefficient (R2) of the calibration curve is 0.9989, and the limit of detection (LOD) and limit of quantification (LOQ) were 0.072 ng/μl and 0.219 ng/μl, respectively. The intra-day and inter-day precision of this analytical method are 2.30–12.80% and 2.93–11.97%, respectively. Recovery was measured as 85.53–101.87% by adding signature peptide with 0.5, 1.0, 7.5 ng/μl into the blank matrix. The method showed no cross-reactivity with squid, tilapia and chicken. Also, the current validated method was successfully applied to shrimps and processed foods. The levels of tropomyosin in seven Taiwanese shrimps ranged from 555.50 to 973.93 μg/g. The present study demonstrated this analytical approach may be an useful technique to analyze tropomyosin in foods.
•ALSNAEGEVAALNR was used as the signature peptide for analysis.•The characteristic fragmentation transition of m/z 708 > 157 were used for tropomyosin quantification.•The LOD and LOQ of this analytical method were 0.072 ng/μl and 0.219 ng/μl, respectively.•Levels of tropomyosin in Taiwanese shrimps were showed in this study.
Protein phosphorylation is a crucial post-translational modification that plays an important role in the regulation of cellular signaling processes. Site-specific quantitation of phosphorylation ...levels can help decipher the physiological functions of phosphorylation modifications under diverse physiological statuses. However, quantitative analysis of protein phosphorylation degrees is still a challenging task due to its dynamic nature and the lack of an internal standard simultaneously available for the samples differently prepared for various phosphorylation extents. In this study, stable-isotope dimethyl labeling coupled with phosphatase dephosphorylation (DM + deP) was tried to determine the site-specific degrees of phosphorylation in proteins. Firstly, quantitation accuracy of the (DM + deP) approach was confirmed using synthetic peptides of various simulated phosphorylation degrees. Afterwards, it was applied to evaluate the phosphorylation stoichiometry of milk caseins. The phosphorylation degree of Ser130 on α-S1-casein was also validated by absolute quantification with the corresponding synthetic phosphorylated and nonphosphorylated peptides under a selected reaction monitoring (SRM) mode. Moreover, this (DM + deP) method was used to detect the phosphorylation degree change of Ser82 on the Hsp27 protein of HepG2 cells caused by
-butyl hydroperoxide (
-BHP) treatment. The results showed that the absolute phosphorylation degree obtained from the (DM + deP) approach was comparable with the relative quantitation resulting from stable-isotope dimethyl labeling coupled with TiO
enrichment. This study suggested that the (DM + deP) approach is promising for absolute quantification of site-specific degrees of phosphorylation in proteins, and it may provide more convincing information than the relative quantification method.
In this study, a novel angiotensin-converting enzyme (ACE)-inhibitory tripeptide (IVR) was isolated and identified from unfertilized soft-shelled turtle egg white (SSTEW). The IC50 value of IVR was ...measured in vitro as low as 0.81 ± 0.03 μM, and its inhibition type was suggested as competitive according to the Lineweaver–Burk plot. This peptide can be generated from either thermolysin followed by trypsin digestion (two stages) or only trypsin digestion (one stage). Quantitative LC–MS/MS analysis indicated that two-stage digestion gave 3.14 ± 0.17 mg of IVR from 1 g of SSTEW, better than that from one-stage digestion (1.31 ± 0.12 mg). In vivo antihypertensive activity of the tripeptide IVR after single oral administration (0.1 and 1 mg/kg of body weight) led to a significant reduction in systolic blood pressure 2–4 h after administration in spontaneously hypertensive rats. In addition, the binding mechanism of IVR has been rationalized through docking simulations using the testicular ACE (tACE)–lisinopril complex at 2 Å resolution (PDB ). The best docking pose was located at the tACE catalytic site resembling the mode of inhibition exerted by lisinopril, an effective hypertensive synthetic drug. The degree of inhibition of this peptide correlated with the H-bond interaction between the C-terminal of IVR and Lys511 and Tyr520 residues of tACE, a significant inhibitor registration for lisinopril. This study illustrated that IVR behaves as a transition-state analogue inhibitor and is useful in therapeutic intervention for blood pressure control. To the best of our knowledge, this is the first report of an efficient ACE-inhibitory tripeptide generated from the unfertilized egg of soft-shelled turtle.
To improve heat dissipation of sapphire-based LEDs, we develop a new LED package with a dual heat spreader design. The first heat spreader is a cup-shaped copper sheet, which was directly contacted ...with sapphire to enhance heat dissipation of the chip itself. The second heat spreader is the die-bonding material of diamond-added AgSnCu solder and a high thermal conductive metal-core printed circuit board (MCPCB), where the conventional dielectric layer was replaced with a thin diamond-like layer. Characterization results demonstrate that the diamond-added composite solder is useful in reducing LED thermal resistance, thus avoiding the thermal accumulation phenomenon. In addition, a LED packaged on the new MCPCB exhibits smaller total thermal resistance and larger light output power.
An A–D–A oligomer, DTS(F2BT)2, was synthesized; its structural evolution was studied with DSC, POM, 2D-WAXD, and in-situ GI-XRD. The structural evolution of DTS(F2BT)2 is stepwise and kinetically ...slow. Both rapid drying and the presence of PC71BM trapped DTS(F2BT)2 in a less ordered nematic (N) phase. PDMS-assisted crystallization enabled a pristine DTS(F2BT)2 thin film to attain a more ordered equilibrium phase, and enhanced the OFET mobility of DTS(F2BT)2. In OPV devices, DIO additive drove the DTS(F2BT)2 domains in the DTS(F2BT)2:PC71BM blended film from the N phase toward the equilibrium phase, and resulted in enhanced OPV performances. These results reveal the slow ordering process of the A–D–A oligomer, and the importance of monitoring the degree of structural evolution of the active thin films in organic optoelectronics.
Protein N-terminal acetylation is one of the most common modifications occurring co- and post-translationally on either eukaryote or prokaryote proteins. However, compared to other protein ...modifications, the physiological role of protein N-terminal acetylation is relatively unclear. To explore the biological functions of protein N-terminal acetylation, a robust and large-scale method for qualitative and quantitative analysis of this modification is required. Enrichment of N(α)-acetylated peptides or depletion of the free N-terminal and internal tryptic peptides prior to analysis by mass spectrometry are necessary based on current technologies. This study demonstrated a simple strong cation exchange (SCX) fractionation method to selectively enrich N(α)-acetylated tryptic peptides via dimethyl labeling without the need for tedious protective labeling and depleting procedures. This method was introduced for the comprehensive analysis of N-terminal acetylated proteins from HepG2 cells. Several hundred N-terminal acetylation sites were readily identified in a single SCX flow-through fraction. Moreover, the N(α)-acetylated peptides of some protein isoforms were simultaneously observed in the SCX flow-through fraction, which indicated that this approach can be utilized to discriminate protein isoforms with very similar full sequences but different N-terminal sequences, such as β-actin/γ-actin, ERK1/ERK2, α-centractin/β-centractin, and ADP/ATP translocase 2 and 3. Compared to other methods, this method is relatively simple and can be directly implemented in a two-dimensional separation (SCX-RP)-mass spectrometry scheme for quantitative N-terminal proteomics using stable-isotope dimethyl labeling.
Mitochondrial Hsp60 (mtHsp60) plays a crucial role in maintaining the proper folding of proteins in the mitochondria. mtHsp60 self-assembles into a ring-shaped heptamer, which can further form a ...double-ring tetradecamer in the presence of ATP and mtHsp10. However, mtHsp60 tends to dissociate in vitro, unlike its prokaryotic homologue, GroEL. The molecular structure of dissociated mtHsp60 and the mechanism behind its dissociation remain unclear. In this study, we demonstrated that
mtHsp60 (EcHsp60) can form a dimeric structure with inactive ATPase activity. The crystal structure of this dimer reveals symmetrical subunit interactions and a rearranged equatorial domain. The α4 helix of each subunit extends and interacts with its adjacent subunit, leading to the disruption of the ATP-binding pocket. Furthermore, an RLK motif in the apical domain contributes to stabilizing the dimeric complex. These structural and biochemical findings provide new insights into the conformational transitions and functional regulation of this ancient chaperonin.
•Analysis of 10 stilbene-type compounds in Ampelopsis brevipedunculata var. hancei.•ACE inhibitors (+)-hopeaphenol and (+)-vitisin A are abundant in the bark.•Correlation of structure, activity and ...abundance.
In this study, we screened 10 resveratrol derivatives isolated from Ampelopsis brevipedunculata var. hancei (Planch.) Rehder (ABH) for angiotensin I converting enzyme (ACE) inhibitory (ACEI) activity. Among these compounds, (+)-hopeaphenol and (+)-vitisin A showed the lowest IC50 values (∼1.5μM) toward ACE. In addition, the compounds’ abundances and distributions in ABH were profiled using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Interestingly, trimers and tetramers of resveratrol were mainly obtained from the bark of ABH when 90% ethanol was used for extraction. This result implies that the antihypertension effect of ABH extract may be mainly contributed by (+)-hopeaphenol (F1) and (+)-vitisin A (F2) in the ABH bark due to their remarkable ACE inhibitions. Moreover, the sizes and structures of these compounds were further correlated to their affinities toward ACE using molecular docking calculations. The results showed that resveratrol tetramers interact with ACE more favorably than other smaller oligomers.
The thermal management of high-power GaN-based light-emitting diodes (LEDs) soldered with Sn-3 wt.%Ag-0.5 wt.%Cu (SAC305) solder and diamond-added SAC305 solder was evaluated. Diamond addition was ...found to significantly reduce the surface temperature and total thermal resistance of the LEDs, revealing that diamond-added SAC305 solder is a promising die-attach material for high-power LED packaging. Interfacial reactions in the LED solder joints were also investigated. The thin Au wetting layer in the chip’s backside metallization was rapidly consumed in the initial stage of reflow, forming an AuSn
4
phase at the interface. Subsequently, the AuSn
4
phase detached from the interface, leading to dewetting of the SAC305 solder from the LED chip. To avoid dewetting, a new backside metallization of LED chips should be developed for SAC305 solder.
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