Protein Samples Research Articles

Effects of the extraction temperatures on the protein contents, gelatin purities, physicochemical properties, and functional properties of tilapia scale gelatins

Herein, the effects of the extraction temperatures (45, 55, 65, 75, and 85 °C) on the protein contents, gelatin purities, physicochemical properties, and functional properties of tilapia scale gelatins were studied. Among these temperatures, 65 °C was the best extraction temperature to obtain good production yield (16.0 % ± 0.3 %), good protein contents (excellent total amino acid composition of 94.20 ± 0.76 g/100 g of gelatin, the highest hydrophobic amino acids percentage of 32.68 ± 0.24 g/100 g of gelatin), appropriate ATR-FTIR spectra peaks (Amides A, B, I, II, and III), high β-sheet percentage (38.2 % ± 1.3 %), the highest purity of the gelatin structures (280, 140, and 125 kDa), the lowest nanoparticle sizes in atomic force microscopy results, the highest water-holding capacity (25.3 % ± 0.9 %), the highest fat-binding capacity (16.9 % ± 0.1 %), high foaming properties (foaming capacity of 151.7 % ± 7.6 % and foaming stability of 145.8 % ± 6.3 %), the lowest interfacial tension (2.1 ± 0.2 mN/m), the lowest emulsifying activity index (16.5 % ± 0.9 %), the highest emulsifying stability index (88.9 % ± 5.1 %), highest emulsion viscosity (1462 ± 17 mPa·s at the rotary speed of 6 rpm), the lowest initial droplet sizes, and lowest emulsion creaming index (6.9 % ± 0.7 %). This work provided a useful guide to choosing extraction temperature for gelatin extraction and a useful theory on the relationship between compositions and properties of a protein sample.

Immune horses rapidly increase antileukoproteinase and lack type I interferon secretion during mucosal innate immune responses against equine herpesvirus type 1.

Equine herpesvirus type 1 (EHV-1) remains a considerable concern in the equine industry, with yearly outbreaks resulting in morbidity, mortality, and economic losses. In addition to its importance in equine health, EHV-1 is a respiratory pathogen and an alphaherpesvirus, and it may serve as a model for other viruses with similar pathogenicity or phylogeny. Large animal models allow the collection of high-volume samples longitudinally, permitting in-depth investigation of immunological processes. This study was performed on bio-banked nasopharyngeal samples from an EHV-1 infection experiment, where clinical outcomes had previously been determined. Matched nucleic acid and protein samples throughout infection permitted longitudinal quantification of the protein or related proteins of selected differentially expressed genes detected during the transcriptomic screen. The results of this manuscript identified novel innate immune pathways of the upper respiratory tract during the first 24 hours of EHV-1 infection, offering a first look at the components of early mucosal immunity that are indicative of protection.

In Situ ATR-FTIR Studies on the β-Sheet Formation of Native and Regenerated Bombyx mori Silk Material in Solution and Its Potential for Drug Releasing Coatings.

Dynamic attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy at both solutions and coatings of a semicrystalline silk material derived from Bombyx mori was applied to monitor the β-sheet conformation, which is known to correlate with silk protein crystallinity. The secondary structure-sensitive Amide I band was analyzed. Two silk protein samples were studied: native-based silk buffer fibroin (NSF) was extracted from silk glands and regenerated silk fibroin (RSF) was extracted from degummed cocoons. Solutions of both NSF and RSF at 2 mg/mL featured low initial β-sheet contents of 5-12%, which further increased to 47-53% after 24 h. RSF and NSF solutions at 23 mg/mL also featured low initial β-sheet contents of 9-10%, which yet only slightly increased to 16-17% after 24 h. Coatings deposited from RSF solutions showed high surface integrity (Q > 99%) after rinsing in mineralized water, enabling interfacial drug delivery applications. RSF coatings were post-treated with either formic acid (FA) or pure methanol (MeOH) vapor to showcase inducibility of crystalline domains in RSF coatings. Such coatings were loaded with the model antibiotic drugs tetracycline (TCL) and streptomycin (STRP), and the sustained release of TCL was followed in contact with (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer. RSF/TCL coatings post-treated with formic acid (FA) vapor followed by methanol (MeOH) vapor showed a significantly lower (52%) initial burst of rather hydrophobic TCL compared to untreated RSF/TCL coatings (72%), while no such significant release difference was observed for hydrophilic STRP. This was rationalized by a specific interaction between nonpolar TCL and hydrophobic crystalline RSF domains.

Integration of AlphaFold with Molecular Dynamics for Efficient Conformational Sampling of Transporter Protein NarK.

Transporter proteins carry their substrate across the cell membrane by changing their conformation. Thus, conformational dynamics are crucial for transport function. However, clarifying the complete transport cycle is challenging even with the current structural biology approach. Molecular dynamics (MD) simulation is a computational approach that can provide the time-resolved conformational dynamics of transporter proteins in atomic details but suffers from a high computational cost. Here, we integrate state-of-the-art protein structure prediction AI, AlphaFold2 (AF2), with MD simulation to reduce the computational cost. Focusing on the transporter protein NarK, we first show that AF2 sampled broad conformations of NarK, including the inward-open, occluded, and outward-open states. We also applied the coevolution-informed mutation in AF2, identifying state-shifting mutations. Then, we show that MD simulations from AF2-generated outward-open conformation, which is experimentally unresolved, captured the essence of the conformational state. We also found that MD simulations from AF2-generated intermediates showed transient dynamics like a transition state connecting two conformational states. This study paves the way for efficient conformational sampling of transporter proteins.

Evaluating the Efficacy of Fecal Immunochemical Test, Fecal Calprotectin, and Serum C-Reactive Protein in Diagnosing Patients With Chronic Lower Gastrointestinal Symptoms.

Accurate early detection of ileocolonic lesions in patients with chronic lower gastrointestinal symptoms (LGISs) is often difficult due to the rarity of early-stage alarm signs. This study assesses the effectiveness of noninvasive blood and stool biomarkers in diagnosing ileocolonic lesions in patients with chronic LGISs undergoing colonoscopy. We conducted a prospective study between April 2020 and July 2022 involving patients with LGISs lasting a month or more. Before colonoscopy, we gathered clinical data, blood samples for C-reactive protein (CRP) and stool samples for fecal immunochemical test (FIT) and fecal calprotectin (FC) analysis. Of 922 participants analyzed (average age 62 years, 37% male), 130 (14.1%) had significant colonoscopy findings, including cancer, advanced adenoma, and inflammatory conditions. Test effectiveness showed an area under the curve of 0.630 for alarm features, 0.643 for CRP, 0.781 for FIT, and 0.667 for FC. Combining stool tests with alarm features improved diagnostic precision. Those without alarm features had a high negative predictive value of 0.97 with low threshold FIT and FC, missing minimal significant lesions, and no cancer. For patients with alarm features, dual high-cutoff test positivity showed a positive predictive value of 0.67. Adding CRP to fecal tests did not enhance accuracy. FIT and FC are valuable in evaluating LGISs. Negative results at low cutoffs can delay colonoscopy in limited resource settings while positive results at dual high cutoffs substantiate the need for the procedure.

The Proteomic Analysis of Chronic Migraine Exosomes Reveals Disease Patterns and Potential Biomarkers.

Exosomes have been identified as optimal biomarkers to screen for multiple diseases. However, few studies focus on the abundant exosome population isolated from plasma of migraine. This study investigated whether proteins in abundant exosomes can aid in the diagnosis of chronic migraine (CM). Plasma exosomes were collected by centrifugation, from which protein samples were extracted. A pilot study (CM, 18; episodic migraine (EM), 26) followed by a second dataset (CM, 26; EM, 16; tension-type headache (TTH), 20; control, 22) was applied to establish a diagnostic model of CM. We employed proteomics based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to search for potential candidate biomarkers in plasma exosomes from CM patients. In total, 530 proteins in plasma exosomes were co-detected. Among them, 13 proteins were found significantly dysregulated between the plasma exosomes of CM patients and other groups. The receiver operating characteristic curve analysis revealed a combination of six proteins (upregulated: RAP2B, AK1, BID, DAG1, PICALM, PSMB2) could distinguish CM patients with high accuracy. Linear correlation analysis showed that the combination was significantly correlated with Headache Impact Test (HIT-6) scores (assessing the negative impact of headaches on normal daily activity). The RT-qPCR results showed the same trends in CM models with nitroglycerin as the exosomal protein sequencing results. These data revealed dysregulated proteins in plasma exosomes of CM, and the combination of plasma exosomal proteins RAP2B, AK1, BID, DAG1, PICALM, and PSMB2 could serve as a novel candidate biomarker for CM diagnosis.

Protein analysis by desorption electrospray ionization mass spectrometry.

This review presents progress made in the ambient analysis of proteins, in particular by desorption electrospray ionization-mass spectrometry (DESI-MS). Related ambient ionization techniques are discussed in comparison to DESI-MS only to illustrate the larger context of protein analysis by ambient ionization mass spectrometry. The review describes early and current approaches for the analysis of undigested proteins, native proteins, tryptic digests, and indirect protein determination through reporter molecules. Applications to mass spectrometry imaging for protein spatial distributions, the identification of posttranslational modifications, determination of binding stoichiometries, and enzymatic transformations are discussed. The analytical capabilities of other ambient ionization techniques such as LESA and nano-DESI currently exceed those of DESI-MS for in situ surface sampling of intact proteins from tissues. This review shows, however, that despite its many limitations, DESI-MS is making valuable contributions to protein analysis. The challenges in sensitivity, spatial resolution, and mass range are surmountable obstacles and further development and improvements to DESI-MS is justified.

6-Plex Tandem Phosphorus Tags (TPT) for Accurate Quantitative Proteomics.

Isobaric chemical labeling is a widely used strategy for high-throughput quantitative proteomics based on mass spectrometry. However, commercially available reagents have high costs in applications as well as the sensitivity limitations for detection of the trace protein samples. Previously, we developed a 2-plex isobaric labeling strategy based on phosphorus chemistry for ultrasensitive proteome quantification with high accuracy. In this work, 6-plex tandem phosphorus tags (TPT) were developed with 3-fold increase in the multiplexing quantitative capacity compared to the 2-plex isobaric phosphorus reagents introduced previously. High isotope enrichment of 18O labeling was incorporated into the phosphoryl group with three exchangeable oxygen atoms by using commercially available H218O. The combinational incorporations of 18O atom in reporter ions and balance group set up the low-cost foundation for development of multiplex TPT reagents. The novel 6-plex TPT reagents could produce phosphoramidate as unique reporter ions with approximately 1 Da mass difference and thus enable 6-plex quantitative analysis in high-resolution ESI-MS/MS analysis. Using HeLa cell tryptic peptides, we concluded that 6-plex TPT reagents could facilitate large-scale accurate quantitative proteomics with very high labeling efficiency.

A Proteogenomic Pipeline for the Analysis of Protein Biosynthesis Errors in the Human Pathogen Candida albicans

Candida albicans is a diploid pathogen known for its ability to live as a commensal fungus in healthy individuals but causing both superficial infections and disseminated candidiasis in immunocompromised patients where it is associated with high morbidity and mortality. Its success in colonizing the human host is attributed to a wide range of virulence traits that modulate interactions between the host and the pathogen, such as optimal growth rate at 37°C, the ability to switch between yeast and hyphal forms, and a remarkable genomic and phenotypic plasticity. A fascinating aspect of its biology is a prominent heterogeneous proteome that arises from frequent genomic rearrangements, high allelic variation, and high levels of amino acid misincorporations in proteins. This leads to increased morphological and physiological phenotypic diversity of high adaptive potential, but the scope of such protein mistranslation is poorly understood due to technical difficulties in detecting and quantifying amino acid misincorporation events in complex protein samples. We have developed and optimized mass spectrometry and bioinformatics pipelines capable of identifying rare amino acid misincorporation events at the proteome level. We have also analyzed the proteomic profile of an engineered C.albicans strain that exhibits high level of leucine misincorporation at protein CUG sites and employed an invivo quantitative gain-of-function fluorescence reporter system to validate our LC-MS/MS data. C.albicans misincorporates amino acids above the background level at protein sites of diverse codons, particularly at CUG, confirming our previous data on the quantification of leucine incorporation at single CUG sites of recombinant reporter proteins, but increasing misincorporation of Leucine at these sites does not alter the translational fidelity of the other codons. These findings indicate that the C.albicans statistical proteome exceeds prior estimates, suggesting that its highly plastic phenome may also be modulated by environmental factors due to translational ambiguity.

Upcycling of Defatted Sesame Seed Meal via Protein Amyloid-Based Nanostructures: Preparation, Characterization, and Functional and Antioxidant Attributes.

Herein, the possibility of valorizing defatted sesame seed meal (DSSM) as a viable source for valuable plant proteins and amyloid-based nanostructure was investigated. Sesame seed protein isolate (SSPI) and the major storage protein globulin (SSG) were prepared by alkaline extraction-isoelectric point precipitation as well as fractionation in the case of SSG. The protein samples were characterized for their physicochemical attributes. SSPI and SSG were also evaluated for their ability to form amyloid structures under heating (90 °C) at low pH (2.0). Additionally, the functional attributes, antioxidant activity, and biocompatibility of the proteins and amyloid nanostructures were also examined. SSPI and SSG were both successfully prepared from DSSM. The data showed that the physicochemical attributes of both protein samples were quite similar, except for the fact that SSG was mostly composed of 11S globulin, as evinced by Tricine-SDS-PAGE analysis. TEM micrographs revealed that SSG was able to form curly-shaped fibrillar amyloid structures, whereas those derived from SSPI were mostly amorphous. Thioflavin-T assay and Tricine-SDS-PAGE analysis indicated that acidic heating promoted protein hydrolysis and self-aggregation of the hydrolyzed peptides into a β-sheet rich amyloid structure. Importantly, the amyloid preparations displayed commendable solubility, superior water and oil holding capacities, and antioxidant activity against DPPH and ABTS. The protein amyloid nanostructures were found to be non-toxic against RAW264.7 cells, HaCaT cells, and red blood cells. These findings indicate that DSSM could be upcycled into valuable protein amyloid structures with good potentialities as novel food ingredients.

Filling the gaps in peptide maps with a platform assay for top-down characterization of purified protein samples.

Liquid chromatography-mass spectrometry (LC-MS) intact mass analysis and LC-MS/MS peptide mapping are decisional assays for developing biological drugs and other commercial protein products. Certain PTM types, such as truncation and oxidation, increase the difficulty of precise proteoform characterization owing to inherent limitations in peptide and intact protein analyses. Top-down MS (TDMS) can resolve this ambiguity via fragmentation of specific proteoforms. We leveraged the strengths of flow-programmed (fp) denaturing online buffer exchange (dOBE) chromatography, including robust automation, relatively high ESI sensitivity, and long MS/MS window time, to support a TDMS platform for industrial protein characterization. We tested data-dependent (DDA) and targeted strategies using 14 different MS/MS scan types featuring combinations of collisional- and electron-based fragmentation as well as proton transfer charge reduction. This large, focused dataset was processed using a new software platform, named TDAcquireX, that improves proteoform characterization through TDMS data aggregation. A DDA-based workflow provided objective identification of αLac truncation proteoforms with a two-termini clipping search. A targeted TDMS workflow facilitated the characterization of αLac oxidation positional isomers. This strategy relied on using sliding window-based fragment ion deconvolution to generate composite proteoform spectral match (cPrSM) results amenable to fragment noise filtering, which is a fundamental enhancement relevant to TDMS applications generally.

Multi-omics analysis reveals that Cas13d contributes to PI3K-AKT signaling and facilitates cell proliferation via PFKFB4 upregulation

The CRISPR-Cas system is a powerful gene editing technology, the clinical application of which is currently constrained due to safety concerns. A substantial body of safety research concerning Cas9 exists; however, scant attention has been directed toward investigating the safety profile of the emergent Cas13 system, which confers RNA editing capabilities. In particular, uncertainties persist regarding the potential cellular impacts of Cas13d in the absence of reliance on a cleavage effect. In this study, we conducted an initial exploration of the effects of Cas13d on HeLa cells. Total RNA and protein samples were extracted after transfection with a Cas13d-expressing plasmid construct, followed by transcriptomic and proteomic sequencing. Differential gene expression analysis identified 94 upregulated and 847 downregulated genes, while differential protein expression analysis identified 185 upregulated and 231 downregulated proteins. Subsequently, enrichment analysis was conducted on the transcriptome and proteome sequencing data, revealing that the PI3K-Akt signaling pathway is a common term. After intersecting the differentially expressed genes enriched in the PI3K-Akt signaling pathway with all the differentially expressed proteins, it was found that the expression of the related regulatory gene PFKFB4 was upregulated. Moreover, western blot analysis demonstrated that Cas13d can mediate PI3K-Akt signaling upregulation through overexpression of PFKFB4. CCK-8 assay, colony formation, and EdU experiments showed that Cas13d can promote cell proliferation. Our data demonstrate, for the first time, that Cas13d significantly impacts the transcriptomic and proteomic profiles, and proliferation phenotype, of HeLa cells, thus offering novel insights into safety considerations regarding gene editing systems.

Fixed-target pump-probe SFX: eliminating the scourge of light contamination.

X-ray free-electron laser (XFEL) light sources have enabled the rapid growth of time-resolved structural experiments, which provide crucial information on the function of macromolecules and their mechanisms. Here, the aim was to commission the SwissMX fixed-target sample-delivery system at the SwissFEL Cristallina experimental station using the PSI-developed micro-structured polymer (MISP) chip for pump-probe time-resolved experiments. To characterize the system, crystals of the light-sensitive protein light-oxygen-voltage domain 1 (LOV1) from Chlamydomonas reinhardtii were used. Using different experimental settings, the accidental illumination, referred to as light contamination, of crystals mounted in wells adjacent to those illuminated by the pump laser was examined. It was crucial to control the light scattering from and through the solid supports otherwise significant contamination occurred. However, the results here show that the opaque MISP chips are suitable for defined pump-probe studies of a light-sensitive protein. The experiment also probed the sub-millisecond structural dynamics of LOV1 and indicated that at Δt = 10 µs a covalent thioether bond is established between reactive Cys57 and its flavin mononucleotide cofactor. This experiment validates the crystals to be suitable for in-depth follow-up studies of this still poorly understood signal-transduction mechanism. Importantly, the fixed-target delivery system also permitted a tenfold reduction in protein sample consumption compared with the more common high-viscosity extrusion-based delivery system. This development creates the prospect of an increase in XFEL project throughput for the field.

Ultrasonic treatment assisted extraction of sunflower protein

Sunflower meal is a promising source of protein, which can be used as a food ingredient in the formulations of meat, flour and confectionery products. Alkaline extraction technology is traditionally used to obtain sunflower protein, but its efficiency can be increased using physical methods: ultrasound, microwave radiation, extraction at high pressure and others. Thу article deals with an application of the ultrasonic treatment for increase of the efficiency of protein extraction from defatted plant material: from the protein fraction of sunflower meal and from sunflower meal. The trial samples for protein extraction were prepared by the preliminary treatment of the samples in the ultrasonic bath for 15 minutes at a frequency of 40 hertz at temperature 24-28° C. The control sample was not subjected to such pretreatment. Then protein was isolated from the raw material by alkaline extraction followed by isoelectric precipitation. The possibility of the protein preparation isolation with a higher crude protein content (93,66% m.f.b) compared to the control sample has been shown. The mass yield of protein was 64% of its content in the raw material. The influence of ultrasonic treatment on the efficiency of protein extraction from raw material with different crude protein content is demonstrated. The results of the study show the feasibility of usage of ultrasound treatment in isolation of sunflower protein. In particular, the crude protein content in the trial protein sample after ultrasonic treatment was increased by 8,23% compared to the control sample. The obtained results demonstrated their consistency when compared to the results of other studies. However, there are only a limited number of studies on the use of ultrasound in the extraction of sunflower derived products.A comparison of the results obtained showed their consistency with the results of other studies. However, there are only a small number of studies on the use of ultrasound in the extraction of sunflower processing products.

Monitoring of the disulfide scrambled species by mixed-mode SEC-HPLC during the purification of a bispecific antibody

Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is an analytical method routinely used for assessing aggregation content in protein samples. As SEC-HPLC separates analytes based on their hydrodynamic radius, it generally lacks the capability of differentiating species that are similar in size. Recently while purifying a bispecific antibody (bsAb), we noticed that SEC-HPLC can provide certain degree of resolution between the target bsAb and a disulfide scrambled form, although these two species were identical in molecular weight. In seeing the unexpected potential of SEC-HPLC at resolving species with similar size, we further tested Zenix SEC-300, a mixed-mode SEC-HPLC column from Sepax, which was reported to be capable of separating protein analytes based on other factors besides size. The Zenix column indeed provided resolution much better than the regular SEC-HPLC column. Upon further optimization, the Zenix column allowed close to baseline separation of the correctly folded and the disulfide scrambled species. The current study, as a complement to the previous reports, further demonstrates that mix-mode SEC-HPLC is capable of separating protein analytes that are close in size but are different in conformation and/or surface characteristics.

Data-Independent Acquisition Represents a Promising Alternative for Fast Photochemical Oxidation of Proteins (FPOP) Samples Analysis.

Fast Photochemical Oxidation of Proteins (FPOP) is a protein footprinting method utilizing hydroxyl radicals to provide valuable information on the solvent-accessible surface area. The extensive number of oxidative modifications that are created by FPOP is both advantageous, leading to great spatial resolution, and challenging, increasing the complexity of data processing. The precise localization of the modification together with the appropriate reproducibility is crucial to obtain relevant structural information. In this paper, we propose a novel approach combining validated spectral libraries together with utilizing DIA data. First, the DDA data searched by FragPipe are subsequently validated using Skyline software to form a spectral library. This library is then matched against the DIA data to filter out nonrepresentative IDs. In comparison with FPOP data processing using only a search engine followed by generally applied filtration steps, the manually validated spectral library offers higher confidence in identifications and increased spatial resolution. Furthermore, the reproducibility of quantification was compared for DIA, DDA, and MS-only acquisition modes on timsTOF SCP. Comparison of coefficients of variation (CV) showed that the DIA and MS acquisition modes exhibit significantly better reproducibility in quantification (CV medians 0.1233 and 0.1494, respectively) compared to the DDA mode (CV median 0.2104).

Probing High-order Protein Complexes Using Native Mass Spectrometry and Hydrogen/Deuterium Exchange Mass Spectrometry: A Case Study Using Fresh and Commercial Hemoglobin Samples.

Native mass spectrometry (MS) analysis of protein complexes is highly susceptible to matrix effect, and addressing this predicament using buffer exchange is a common approach. Nevertheless, optimization of the buffer exchange protocol is not trivial. With the use of hemoglobin (Hb) as the model entity, it was discovered that the native mass spectrum of protein assembly is highly dependent on the buffer-exchange protocol. Given the dependence of native MS on the purification protocol, this work attempts to use hydrogen/deuterium exchange mass spectrometry (HDX-MS) for comparative studies of hemoglobin complexes in untreated fresh and commercial samples. The information obtained from the HDX study was found to correlate well with the native mass spectrometry analysis of the properly buffer-exchanged Hb samples. Both native MS and HDX-MS showed that the fresh Hb sample has retained the expected tetrameric structure, whereas the commercial Hb has largely been denatured to the dimeric form. These findings prove the complementarity of native MS and HDX-MS in the analysis of high-order protein complexes and stress the necessity to validate the integrity of the high-order structures of the proteins prior to the use of the protein samples for other biomedical studies.

CryoCycle your grids: Plunge vitrifying and reusing clipped grids to advance cryoEM democratization.

CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods, called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples. Furthermore, we demonstrate the vitrification of thin areas of cells prepared on gold-coated, pre-clipped grids.

Freezing Diluted Bovine Serum Albumin Standards Does Not Significantly Affect Standard Curves.

Total protein isolation followed by quantitation is a common protocol in many laboratories. Quantitation is often done using a colorimetric assay such as the bicinchoninic acid (BCA) assay in which a change in the color of the BCA reagent is related to protein concentration. Extracted protein samples are compared to a standard curve made with dilutions of a protein standard such as bovine serum albumin (BSA) to determine their concentrations. A series of experiments was designed to determine the most reproducible and accurate method for quantifying protein concentrations of samples in an experimental series over time. The effect of freezing on diluted standards was investigated. Standards were frozen at -20°C or -80°C and serially thawed and refrozen up to three times prior to their use in a BCA assay. Thawing and refreezing the standards had no significant effect on protein concentration and the resulting standard curves. Inter-person and intra-person variability in the preparation of standards was also investigated. Protein concentration differences due to inter-person and intra-person variability were greater than protein concentration variability resulting from freezing and thawing, regardless of the freezing temperature. The most reproducible and accurate method for determining the protein concentration of extracted samples in an experimental series over time is diluting a large batch of BSA standards and freezing them at either -20°C or -80°C. Reproducibility was maintained with up to three freeze-thaws.

The Effect of β-Sheet Secondary Structure on All-β Proteins by Molecular Dynamics Simulations.

The effect of β-sheet ratio and chain length on all-β proteins was investigated by MD simulations. Protein samples composed of different repeating units with various β-sheet ratios or a different number of repeating units were simulated under a broad temperature range. The simulation results show that the smaller radius of gyration was achieved by the protein with the higher proportion of β-sheet secondary structure, which had the lower nonbonded energy with more HBs within the protein. The root mean square deviation (RMSD) and the root mean square fluctuation (RMSF) both increased with temperature, especially in the case of a longer chain. The visible period was also shown according to the repeated secondary structure. Several minimum values of RMSF were located on the skeleton of Cα atoms participating in the β-sheet, indicating that it is a kind of stable secondary structure. We also concluded that proteins with a short chain or a lower ratio of β-sheet could easily transform their oriented and compact structures to other ones, such as random coils, turns, and even α-helices. These results clarified the relationship from the primary level to the 3D structure of proteins and potentially predicted protein folding.