Combining SDS-PAGE to capillary zone electrophoresis-tandem mass spectrometry for high-resolution top-down proteomics analysis of intact histone proteoforms.

Mass spectrometry (MS)-based top-down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post-translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high-resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS-PAGE-based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI-MS) with capillary zone electrophoresis (CZE)-MS/MS for high-resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS-PAGE gel and follow-up cleanup as well as CZE-MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high-resolution separation and characterization of histone proteoforms. SDS-PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high-resolution separations of SDS-PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.

Residual protein analysis by SDS-PAGE in clinically manufactured BM-MSC products.

Residual substances that are considered hazardous to the recipient must be removed from final cellular therapeutic products manufactured for clinical purposes. In doing so, quality rules determined by competent authorities (CAs) for the clinical use of tissue- and cell-based products can be met. In our study, we carried out residual substance analyses, and purity determination studies of trypsin and trypsin inhibitor in clinically manufactured bone marrow-derived mesenchymal stromal/stem cell products, using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. Despite being a semiquantitative method, SDS-PAGE has several benefits over other methods for protein analysis, such as simplicity, convenience of use, and affordability. Due to its convenience and adaptability, SDS-PAGE is still a commonly used method in many laboratories, despite its limits in dynamic range and quantitative precision. Our goal in this work was to show that SDS-PAGE may be used effectively for protein measurement, especially where practicality and affordability are the major factors. The results of our study suggest a validated method to guide tissue and cell manufacturing sites for making use of an agreeable, accessible, and cost-effective method for residual substance analyses in clinically manufactured cellular therapies.

Growth and protein response of rice plant with plant growth-promoting rhizobacteria inoculations under salt stress conditions.

Soil salinity has been one of the significant barriers to improving rice production and quality. According to reports, Bacillus spp. can be utilized to boost plant development in saline soil, although the molecular mechanisms behind the interaction of microbes towards salt stress are not fully known. Variations in rice plant protein expression in response to salt stress and plant growth-promoting rhizobacteria (PGPR) inoculations were investigated using a proteomic method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Findings revealed that 54 salt-responsive proteins were identified by mass spectrometry analysis (LC-MS/MS) with the Bacillus spp. interaction, and the proteins were functionally classified as gene ontology. The initial study showed that all proteins were labeled by mass spectrometry analysis (LC-MS/MS) with Bacillus spp. interaction; the proteins were functionally classified into six groups. Approximately 18 identified proteins (up-regulated, 13; down-regulated, 5) were involved in the photosynthetic process. An increase in the expression of eight up-regulated and two down-regulated proteins in protein synthesis known as chaperones, such as the 60 kDa chaperonin, the 70 kDa heat shock protein BIP, and calreticulin, was involved in rice plant stress tolerance. Several proteins involved in protein metabolism and signaling pathways also experienced significant changes in their expression. The results revealed that phytohormones regulated the manifestation of various chaperones and protein abundance and that protein synthesis played a significant role in regulating salt stress. This study also described how chaperones regulate rice salt stress, their different subcellular localizations, and the activity of chaperones.

Characterization of heat, salt, acid, alkaline, and antibiotic stress response in soil isolate Bacillus subtilis strain PSK.A2.

Microbes play an essential role in soil fertility by replenishing the nutrients; they encounter various biotic and abiotic stresses disrupting their cellular homeostasis, which expedites activating a conserved signaling pathway for transient over-expression of heat shock proteins (HSPs). In the present study, a versatile soil bacterium Bacillus subtilis strain PSK.A2 was isolated and characterized. Further, the isolated bacterium was exposed with several stresses, viz., heat, salt, acid, alkaline, and antibiotics. Stress-attributed cellular morphological modifications such as swelling, shrinkage, and clump formation were observed under the scanning electron microscope. The comparative protein expression pattern was studied by SDS-PAGE, relative protein stabilization was assessed by protein aggregation assay, and relative survival was mapped by single spot dilution and colony-counting method under control, stressed, lethal, and stressed lethal conditions of the isolate. The findings demonstrated that bacterial stress tolerance was maintained via the activation of various HSPs of molecular weight ranging from 17 to 115 kD to respective stimuli. The treatment of subinhibitory dose of antibiotics not interfering protein synthesis (amoxicillin and ciprofloxacin) resulted in the expression of eight HSPs of molecular weight ranging from 18 to 71 kD. The pre-treatment of short stress dosage showed endured overall tolerance of bacterium to lethal conditions, as evidenced by moderately enhanced total soluble intracellular protein content, better protein stabilization, comparatively over-expressed HSPs, and relatively enhanced cell survival. These findings hold an opportunity for developing novel approaches towards enhancing microbial resilience in a variety of conditions, including industrial bioprocessing, environmental remediation, and infectious disease management.

Bioproduction, purification, partial characterization and phenol removal efficacy of tyrosinase enzyme from Streptomyces sp. strain MR28.

The study focused on the production of the tyrosinase enzyme from Streptomyces sp. MR28 and its potency in removal of phenol content from water using free and immobilized tyrosinase enzyme. The tyrosinase was produced by Streptomyces sp. MR28 in liquid tyrosine broth medium, and it was further purified to near its homogeneity by employing, precipitation, dialysis, and column chromatography. After the purification, 44.49% yield with a 4 fold purification was achieved. The characterization of the purified enzyme showed a single major peak on HPLC and a solitary band on SDS-PAGE. The purified tyrosinase enzyme was active at a pH of 7.0 and a temperature of 30 °C. Further immobilization of purified tyrosinase was performed using the sodium alginate entrapment method. The capacity of the purified tyrosinase to remove phenol in water was evaluated by spectrophotometric method. The free tyrosinase enzyme-treated solutions showed a gradual decrease in the concentration of phenol with increased incubation time at 30 °C and 40 °C, at 90 min of the incubation time, it showed maximum efficacy in removing phenol from the solution. At 50 °C and 60 °C, the free tyrosinase enzyme exhibited very less capacity to remove the phenol. The immobilized enzyme showed good capacity for the removal of phenol from the solutions; the concentration of phenol in the solution decreased with an increase in the incubation time. At temperatures of 40 °C and 50 °C, the immobilized tyrosinase enzyme beads showed significant removal of phenol from the solution, and at temperatures of 30 °C and 60 °C, they also exhibited good capacity for the removal of phenol. At the end of the 90 min incubation period, it exhibited good capability. The current study suggests using immobilized microbial tyrosinase enzyme can be used for the removal of phenol from the contaminated water in a greener manner.

Impact of Ultrafiltration on the Physicochemical Properties of Bovine Lactoferrin: Insights into Molecular Mass, Surface Morphology, and Elemental Composition.

The large-scale isolation of bovine lactoferrin (bLF) typically involves using large amounts of concentrated eluents, which might introduce impurities to the final product. Sometimes, protein pre-concentration is required for the greater accuracy of experimental results. In this research, the supplied bLF sample was subjected to additional ultrafiltration (UF) to eliminate possible small impurities, such as salts and peptides of bLF. Beforehand, the basic characterization of native bLF, including surface-charge properties and the structural sensitivity to the various pH conditions, was performed. The study aimed to evaluate the difference in molecular mass, primary structure, surface morphology, and elemental composition of the protein before and after UF. The research was provided by application of spectroscopic, spectrometric, electrophoretic, and microscopic techniques. The evident changes in the surface morphology of bLF were observed after UF, while the molecular masses of both proteins were comparable. According to MALDI-TOF/MS results, UF had a positive impact on the bLF sample representation, improving the identification parameters, such as sequence coverage and intensity coverage.

Spray drying and ultrasonication processing of camel whey protein concentrate: Characterization and impact on bioactive properties.

The production of whey protein concentrates (WPCs) from camel milk whey represents an effective approach to valorize this processing by-product. These concentrates harbor active ingredients with significant bioactive properties. Camel WPCs were spray-dried (SD) at inlet temperature of 170, 185 and 200°C, or Ultrasonicated (US) for 5, 10 and 15 min, then freeze-dried to obtain fine powder. The impact of both treatments on protein degradation was studied by sodium dodecyl sulfate-PAGE and reverse-phase ultraperformance liquid chromatography (RP-UPLC) techniques. Significantly enhanced protein degradation was observed after US treatment when compared with SD. Both SD and US treatments slightly enhanced the WPCs samples' antioxidant activities. The US exposure for 15 min exhibited highest 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity (12.12 mmol TE/g). Moreover, US treatment for 10 min exhibited the highest in vitro anti-diabetic properties (α-amylase and α-glucosidase inhibition), and dipeptidyl-peptidase-IV inhibitory activity among all samples. In addition, the ultrasonication for 10 min and SD at 170°C showed the lowest IC50 values for in vitro anti-hypercholesterolemic activities in terms of pancreatic lipase and cholesteryl esterase inhibition. Conclusively, these green techniques can be adapted in the preservation and processing of camel milk whey into active ingredients with high bioactive properties.

Reversal of Valproate-Induced Major Salivary Gland Changes By Moringa Oleifera Extract in Rats.

This study aimed to explore the potential protective impacts of Moringa oleifera extract on major alteration in salivary glands of rats exposed to sodium valproate (VA). Groups were defined as control, control+moringa extract, sodium valproate, and sodium valproate+moringa extract. Antioxidant and oxidant status, activities of digestive and metabolic enzymes were examined. VA treatment led to various biochemical changes in the salivary glands, including decreased levels of antioxidants like glutathione, glutathione-S-transferase, and superoxide dismutase (except for sublingual superoxide dismutase). Conversely, a decrease in alpha-amylase, alkaline and acid phosphatase, lactate dehydrogenase, protease, and maltase activities were observed. The study also demonstrated that VA induces oxidative stress, increases lipid peroxidation, sialic acid, and nitric oxide levels in the salivary glands. Total oxidant capacity was raised in all glands except in the sublingual gland. The electrophoretic patterns of proteins were similar. Moringa oleifera extract exhibited protective properties, reversing these VA-induced biochemical changes due to its antioxidant and therapeutic attributes. This research suggests that moringa extract might serve as an alternative treatment approach for individuals using VA and experiencing salivary gland issues, although further research is necessary to confirm these findings in human subjects.

Whey protein dynamics in goat mammary secretions during colostrum and early lactation periods.

The protein composition in goat milk undergoes changes throughout the different lactation periods, displaying distinct characteristics that are influenced by the dynamic nature of protein composition and concentration during the transition from colostrum secretion to mature milk. To evaluate the dynamics of whey proteins of Saanen goats during the colostral phase and the first month of lactation, 110 milk samples from 11 healthy mammary halves of seven Saanen goats were selected through a clinical evaluation. Whey was obtained by rennet coagulation of the mammary secretion. The biuret method determined total protein concentration, and their fractions were identified by 12% dodecyl sulfate-polyacrylamide gel electrophoresis. Maximum concentrations of all protein fractions were observed in the first 12 h of lactation, reducing throughout the study. Modification of the protein predominance was also observed. The transition from colostrum secretion to milk occurred 5 or 7 d postpartum.

Investigation of biological activities of two cultivars of Cicer arietinum proteins mass associated with Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, with some known classical factors. Cicer arietinum (Leguminosae) is a source of protein for humans and contains albumin, globulin, glutelin, and prolamin. The protein content of two cultivars of C. arietinum, Hashem and Mansour, was isolated to evaluate their inhibition activity against acetylcholinesterase (AChE), butyrylcholine esterase (BChE), and ß-amyloid peptide (ßA) aggregation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and molecular docking were also applied to evaluate the content and determine the potential of each chickpea protein to interact with AChE, respectively. Obtained data showed that proteins from both cultivars could inhibit AChE with IC50 of 17.73 (0.03) and 22.20 (0.06) µg/mL, respectively, with no activity on BChE. The 50 µg/mL protein concentration of each cultivar suppressed ßA accumulation (Mansour: 25.66% and Hashem: 21.69%) and showed biometal chelating activity. SDS-PAGE analysis revealed relatively different protein patterns, though the Mansour cultivar contained some protein bands with molecular weights of 18, 24, and 70 kDa were estimated to belong to vicilin and legumin, which were absent in the Hashem protein mass. Molecular docking showed that legumin and especially vicilin have good potential to interact with AChE. The chickpea proteins showed inhibitory activity against AChE, which might be due to the vicilin and legumin fractions. The characterization of the inhibitory effect of each protein band could be promising in finding new therapeutic peptide candidates to treat Alzheimer's in the future, although more experimental work is needed in this issue.

Development and Optimization of Label-Free Quantitative Proteomics under Different Crossing Periods of Bottle Gourd.

Bottle gourd, a common vegetable in the human diet, has been valued for its medicinal and energetic properties. In this experiment, the time-resolved analysis of the changes in the proteins' electrophoretic patterning of the seed development at different crossing periods was studied in bottle gourd using label-free quantitative proteomics. Hybrid HBGH-35 had the highest observed protein levels at the 4th week of the crossing period (F4) compared to the parental lines, viz. G-2 (M) and Pusa Naveen (F). The crossing period is significantly correlated with grain filling and reserve accumulation. The observed protein expression profile after storage was related to seed maturation and grain filling in bottle gourds. A total of 2517 proteins were identified in differentially treated bottle gourd fruits, and 372 proteins were differentially expressed between different crossing periods. Proteins related to carbohydrate and energy metabolism, anthocyanin biosynthesis, cell stress response, and fruit firmness were characterized and quantified. Some proteins were involved in the development, while others were engaged in desiccation and the early grain-filling stage. F4 was distinguished by an increase in the accumulation of low molecular weight proteins and enzymes such as amylase, a serine protease, and trypsin inhibitors. The seed vigor also followed similar patterns of differential expression of seed storage proteins. Our findings defined a new window during seed production, which showed that at F4, maximum photosynthetic assimilates accumulated, resulting in an enhanced source-sink relationship and improved seed production. Our study attempts to observe the protein expression profiling pattern under different crossing periods using label-free quantitative proteomics in bottle gourd. It will facilitate future detailed investigation of the protein associated with quality traits and the agronomic importance of bottle gourd through selective breeding programs.

The role of native cysteine residues in the oligomerization of KCNQ1 channels.

KCNQ1, the major component of the slow-delayed rectifier potassium channel, is responsible for repolarization of cardiac action potential. Mutations in this channel can lead to a variety of diseases, most notably long QT syndrome. It is currently unknown how many of these mutations change channel function and structure on a molecular level. Since tetramerization is key to proper function and structure of the channel, it is likely that mutations modify the stability of KCNQ1 oligomers. Presently, the C-terminal domain of KCNQ1 has been noted as the driving force for oligomer formation. However, truncated versions of this protein lacking the C-terminal domain still tetramerize. Therefore, we explored the role of native cysteine residues in a truncated construct of human KCNQ1, amino acids 100-370, by blocking potential interactions of cysteines with a nitroxide based spin label. Mobility of the spin labels was investigated with continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. The oligomerization state was examined by gel electrophoresis. The data provide information on tetramerization of human KCNQ1 without the C-terminal domain. Specifically, how blocking the side chains of native cysteines residues reduces oligomerization. A better understanding of tetramer formation could provide improved understanding of the molecular etiology of long QT syndrome and other diseases related to KCNQ1.

The association between hordein polypeptide banding and agronomic traits in partitioning genetic diversity in six-rowed Ethiopian barley lines (Hordeum vulgare L.).

BACKGROUND: Evaluation of the extent of genetic variation within and between the populations of crop genetic resources are of paramount importance in any breeding program. An experiment aimed at assessing the extent of variation among barley lines and the degree of association between hordein polypeptide and agronomic traits was hence executed. METHODS: Field experiment was conducted in six environments between 2017-2019 involving 19 barley lines. Hordein bands were separated using vertical Sodium Dodecyl Sulphate Poly- acrylamide Gel Electrophoresis (SDS-PAGE). RESULTS: The analysis of variance revealed significant variation among lines and wider range units were observed for the agronomic traits. The line (Acc# 16,811-6) was superior, producing the highest grain yield (2.97 ton ha-1) across environments, 3.6 ton ha-1 at Holleta, and 1.93 ton ha-1 at Chefedonsa. At Arsi Negelle a different line Acc# 17146-9 was the highest yielding (3.15ton ha-1). SDS-PAGE-based analysis of barley lines separated 12 hordein bands between C (four bands) and B (eight bands) subunits. Interestingly bands 52, 46a, and 46b were uniquely conserved in the four naked barley lines (Acc#16809-14,16956-11, 17240-3, 17244-19). A considerably high proportion of genetic diversity within the populations than among the populations could be a repercussion of high gene flow which substantiates the longstanding and dominant informal seed exchange system among the farmers. The significant positive association between grain yield and band 50 evocates the expression of this allele may code for higher grain yield. The negative association between days to maturity and band 52 perhaps stipulates earliness in barely lines upon the manifestation of the band. Band 52 and 60 appeared to be associated with more than one agronomic trait (days to maturity and thousand kernel weight; grain filling period and grain yield respectively) and could be the result of pleiotropic characteristics of the genes residing in these banding regions. CONCLUSION: The barley lines exhibited substantial variation for hordein protein and agronomic traits. However, imparted the need for the implementation of decentralized breeding as a consequence of genotype-by-environment interaction. Significant hordein polypeptide and agronomic traits association advocated the utilization of hordein as a protein marker and perhaps consider them in the parental line selection.

Agro-morphological, biochemical, and molecular markers of barley genotypes grown under salinity stress conditions.

The aim of this study was to evaluate the impact of salt stress on morphological, yield, biochemical, and molecular attributes of different barley genotypes. Ten genotypes were cultivated at Fayoum Research Station, El-Fayoum Governorate, Egypt, during two seasons (2020-2021 and 2021-2022), and they were exposed to two different salt concentrations (tap water as a control and 8000 ppm). The results showed that genotypes and salt stress had a significant impact on all morphological and physiological parameters. The morphological parameters (plant height) and yield attributes (spike length, number of grains per spike, and grain yield per plant) of all barley genotypes were significantly decreased under salt stress as compared to control plants. Under salt stress, the total soluble sugars, proline, total phenol, total flavonoid, ascorbic acid, malondialdehyde, hydrogen peroxide, and sodium contents of the shoots of all barley genotypes significantly increased while the potassium content decreased. L1, which is considered a sensitive genotype was more affected by salinity stress than the tolerance genotypes L4, L6, L9, and Giza 138. SDS-PAGE of seed proteins demonstrated high levels of genetic variety with a polymorphism rate of 42.11%. All genotypes evaluated revealed significant variations in the seed protein biochemical markers, with new protein bands appearing and other protein bands disappearing in the protein patterns of genotypes cultivated under various conditions. Two molecular marker techniques (SCoT and ISSR primers) were used in this study. Ten Start Codon Targeted (SCoT) primers exhibited a total of 94 fragments with sizes ranging from 1800 base pairs to 100 base pairs; 29 of them were monomorphic, and 65 bands, with a polymorphism of 62.18%, were polymorphic. These bands contained 21 unique bands (9 positive specific markers and 12 negative specific markers). A total of 54 amplified bands with molecular sizes ranging from 2200 to 200 bp were produced using seven Inter Simple Sequence Repeat (ISSR) primers; 31 of them were monomorphic bands and 23 polymorphic bands had a 40.9% polymorphism. The techniques identified molecular genetic markers associated with salt tolerance in barley crop and successfully marked each genotype with distinct bands. The ten genotypes were sorted into two main groups by the unweighted pair group method of arithmetic averages (UPGMA) cluster analysis based on molecular markers and data at a genetic similarity coefficient level of 0.71.

Germplasm characterization and SDS-PAGE analysis of caper (Capparis spinosa L.) from different provenances.

BACKGROUND: Capparis spinosa L. is a typical desert plant that is resistant to high temperatures and drought, and at the same time is rich in medicinal and food values. The objective of this study is to explore the variations in nutrient composition, morphological characteristics, and SDS-PAGE patterns of caper seeds from different provenances, aiming to provide insights for the selection of superior seed provenances. RESULTS: In this experiment, there were significant differences in the morphological characteristics and major nutritional components of caper seeds from different provenances. Seeds from the YKL (Karayagaqi Township, Yining County) and YKG (G218, KashiTown, Yining County) regions were larger in size compared to seeds from other regions. Among the four measured nutritional components, crude fat had the highest content, especially in the YKL and YKG region. The results of correlation analysis showed that crude fat was negatively correlated with soluble sugar and soluble protein but significantly positively correlated with starch content. As longitude increased from east to west, the morphological characteristics gradually increased. Based on the principal component analysis of all the parameters of the seeds, the eight provenances could be classified into three groups. HM (Hami), TGS (S202, Gaochang District, Turpan), HYW (Wubao Town, Yizhou District, Hami), TQQ (Qiquanhu Town, Turpan), and TLF (Turpan) were a group with higher soluble protein, soluble sugar, and water content. YKL and YKG were in one group, which had larger seed grains with high crude fat and starch content. AKS (Aksu) was in a separate group. The protein fractions from seeds of eight regions were extracted using Osborne fractionation method, it was found that glutelin content was the highest, while albumin content was the lowest. After these proteins were analyzed by SDS-PAGE, the electrophoretic patterns showed that the protein molecular weights were relatively small, and there were differences in protein bands among different provenances. CONCLUSION: According to the PCA results, the eight seed provenances could be divided into three groups. There were both geographically distant ones clustered into one group, and those close to each other were also divided into one group. There were differences in seed morphology, nutrient content and SDS-PAGE profiles among the different seed sources. This difference might be caused by a combination of geographic and climatic factors. In addition, YKL and YKG were roughly selected as good seed provenances, which provided a theoretical basis for the development of C. spinosa L. germplasm resources.

The Internal Structure of the Velvet Worm Projectile Slime: A Small-Angle Scattering Study.

For prey capture and defense, velvet worms eject an adhesive slime which has been established as a model system for recyclable complex liquids. Triggered by mechanical agitation, the liquid bio-adhesive rapidly transitions into solid fibers. In order to understand this mechanoresponsive behavior, here, the nanostructural organization of slime components are studied using small-angle scattering with neutrons and X-rays. The scattering intensities are successfully described with a three-component model accounting for proteins of two dominant molecular weight fractions and nanoscale globules. In contrast to the previous assumption that high molecular weight proteins-the presumed building blocks of the fiber core-are contained in the nanoglobules, it is found that the majority of slime proteins exist freely in solution. Only less than 10% of the slime proteins are contained in the nanoglobules, necessitating a reassessment of their function in fiber formation. Comparing scattering data of slime re-hydrated with light and heavy water reveals that the majority of lipids in slime are contained in the nanoglobules with homogeneous distribution. Vibrating mechanical impact under exclusion of air neither leads to formation of fibers nor alters the bulk structure of slime significantly, suggesting that interfacial phenomena and directional shearing are required for fiber formation.

Speeding up SDS-PAGE: Theory and experiment.

In order to accelerate Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), here we propose an optimized version of the technique enabled by experimental tuning reinforced by theoretical description. In the resulting system, the gel buffer was diluted twofold and supplemented with glycine at a low concentration, whereas a higher voltage was applied. This approach reduced runtime from 90 to 18 min. It is important to emphasize that, despite the high voltage applied to the gel, the resolution of the bands did not decrease compared to the original Laemmli method. The proposed acceleration approach can be used in other variants of SDS-PAGE.

Towards absolute quantification of protein genetic variants in Pisum sativum extracts.

In recent years, several studies have used proteomics approaches to characterize genetic variant profiles of agricultural raw materials. In such studies, the challenge is the quantification of the individual protein variants. In this study a novel UPLC-PDA-MS method with absolute and label-free UV-based peptide quantification was applied to quantify the genetic variants of legumin, vicilin and albumins in pea extracts. The aim was to investigate the applicability of this method and to identify challenges in determining protein concentration from the measured peptide concentrations. Analysis of the protein mass balance showed significant losses of proteins in extraction (37%) and of peptides in further sample preparation (69%). The challenge in calculating the extractable individual protein concentrations was how to deal with these insoluble peptides. The quantification approach using average amino acid concentrations in each position of the sequence showed most reproducible results and allowed comparison of the genetic protein composition of 8 different cultivars. The extractable protein composition (µM/µM) was remarkably similar for all cultivar extracts and consisted of legumins A1 (12.8 ± 1.2%), A2 (1.1 ± 0.4%), B (9.9 ± 1.6%), J (7.5 ± 1.0%) and K (10.3 ± 2.1%), vicilin (15.2 ± 1.7%), provicilin (15.7 ± 2.5%), convicilin (9.8 ± 0.8%), albumin A1 (7.4 ± 2.0%), albumin 2 (10.0 ± 1.5%) and protease inhibitor (0.4 ± 0.4%).

A simple preparation of prestained molecular markers for electrophoresis using inexpensive and readily available proteins.

Protein electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) is one of the most commonly performed procedures in biochemical laboratories. It requires the use of molecular weight (MW) markers as an internal technical control and to determine the migration rate of a particular protein. In this work, we describe a simple method for preparing "homemade" prestained protein markers using readily available cow's milk and chicken egg white proteins without the need of any major protein purification step, and produce prestained MW markers ranging from 19 to 98 kDa.

Comprehensive investigation of a structural variant in a bi-specific, N-and C-terminal Fc-fusion molecule, and its monitoring with LC-MS based method.

There is an increasing interest in the generation of Fc-fusion molecules to exploit the effector functions of Fc and the fusion partner, towards improving the therapeutic potential. The Fc-fusion molecules have unique structural and functional attributes that impart various advantages. However, the manufacturing of Fc-fusion molecules possesses certain challenges in the biopharmaceutical development. The fusion of unnaturally occurring two or more domains in a construct can pose problems for proper folding and are prone to aggregation and degradation. Reshuffling of disulfide bridges represents a posttranslational event that affects folding. This can play a critical role in the correct structure of a molecule and leads to structural heterogeneity in biotherapeutics; it may also impact the in vivo biological activities, safety, and efficacy of the biopharmaceutical. Our work presents an investigation case of a doublet band, as observed only in nonreducing sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) for a bi-specific, N- and C-terminal Fc-fusion molecule. Other characterization and orthogonal methods from the analytical panel did not indicate the presence of two distinct species, including the orthogonal CE-SDS (Caliper Lab Chip GXII). Therefore, it was necessary to determine if the phenomenon was an analytical artifact or a real variant of our Fc-fusion molecule. With the comprehensive mass spectrometry-based characterization, we were able to determine that the doublet band was related to the reshuffling of one disulfide bridge in one of the fused domains. Our work illustrates the application of nonreducing peptide mapping by mass spectrometry to characterize and identify disulfide variants in a complex N- and C-terminal Fc-fusion molecule, and further adoption to monitor the disulfide structural variants in the intermediate process samples to drive the manufacturing of a consistent product with the desired quality attributes.