Fast protein sequencing of monoclonal antibody by real-time digestion on emitter during nanoelectrospray

Growth in the pharmaceutical industry has led to an increasing demand for rapid characterization of therapeutic monoclonal antibodies. The current methods for antibody sequence confirmation (e.g., N-terminal Edman sequencing and traditional peptide mapping methods) are not sufficient; thus, we developed a fast method for sequencing recombinant monoclonal antibodies using a novel digestion-on-emitter technology. Using this method, a monoclonal antibody can be denatured, reduced, digested, and sequenced in less than an hour. High throughput and satisfactory protein sequence coverage were achieved by using a non-specific protease from Aspergillus saitoi, protease XIII, to digest the denatured and reduced monoclonal antibody on an electrospray emitter, while electrospray high voltage was applied to the digestion mixture through the emitter. Tandem mass spectrometry data was acquired over the course of enzyme digestion, generating similar information compared to standard peptide mapping experiments in much less time. We demonstrated that this fast protein sequencing method provided sufficient sequence information for bovine serum albumin and two commercially available monoclonal antibodies, mouse IgG1 MOPC21 and humanized IgG1 NISTmAb. For two monoclonal antibodies, we obtained sequence coverage of 90.5–95.1% for the heavy chains and 98.6–99.1% for the light chains. We found that on-emitter digestion by protease XIII generated peptides of various lengths during the digestion process, which was critical for achieving sufficient sequence coverage. Moreover, we discovered that the enzyme-to-substrate ratio was an important parameter that affects protein sequence coverage. Due to its highly automatable and efficient design, our method offers a major advantage over N-terminal Edman sequencing and traditional peptide mapping methods in the identification of protein sequence, and is capable of meeting an ever-increasing demand for monoclonal antibody sequence confirmation in the biopharmaceutical industry.     

Comparative analysis of microbiota along the length of the gastrointestinal tract of two tree squirrel species (Sciurus aberti and S. niger) living in sympatry

Microbiota inhabiting the gastrointestinal (GI) tract of animals has important impacts on many host physiological processes. Although host diet is a major factor influencing the composition of the gut micro‐organismal community, few comparative studies have considered how differences in diet influence community composition across the length of the GI tract. We used 16S sequencing to compare the microbiota along the length of the GI tract in Abert's (Sciurus aberti) and fox squirrels (S. niger) living in the same habitat. While fox squirrels are generalist omnivores, the diet of Abert's squirrels is unusually high in plant fiber, particularly in winter when they extensively consume fiber‐rich inner bark of ponderosa pine (Pinus ponderosa). Consistent with previous studies, microbiota of the upper GI tract of both species consisted primarily of facultative anaerobes and was less diverse than that of the lower GI tract, which included mainly obligate anaerobes. While we found relatively little differentiation between the species in the microbiota of the upper GI tract, the community composition of the lower GI tract was clearly delineated. Notably, the Abert's squirrel lower GI community was more stable in composition and enriched for microbes that play a role in the degradation of plant fiber. In contrast, overall microbial diversity was higher in fox squirrels. We hypothesize that these disparities reflect differences in diet quality and diet breadth between the species.     

Species composition of semi-natural mesic grasslands as a factor influencing the methane yield of plant biomass (Central Europe)

Semi-natural lowland and mountain mesic meadows are grasslands rich in species, and their conservation status depends on treatments such as mowing or grazing livestock. In many countries, the condition of grasslands is deteriorating because of their inappropriate use or abandonment. This study aimed to determine the effects of the species composition of plant communities and functional plant groups on the methane yield from biomass harvested from mesic grasslands in the Sudetes Mountains. Biogas potential analysis was performed based on biomass samples collected from Poland and the Czech Republic. The biogas potential was determined in 40 day-long batch anaerobic digestion tests. The average methane yield obtained from the biomass was 246 ± 16 NL CH₄ kg^?1 VS, whereas the methane yield per hectare was 870 ± 203 m³ CH₄ ha^?1. Plant communities comprising different dominant species had no effect on the methane yield but affected the methane yield per hectare. Additionally, the species composition of grasslands with a higher percentage of forbs had lower biomass yield, resulting in lower methane yields per hectare. The continuity of the low-intensity management of mountain grassland, which can be provided by the utilization of their biomass for bioenergy production, sustains high biodiversity and ensures appropriate meadow conservation.     

Bioaugmentation of a two-stage thermophilic (68 degrees C/55 degrees C) anaerobic digestion concept for improvement of the methane yield from cattle manure

The possibility of improving a two-stage (68 degrees C/55 degrees C) anaerobic digestion concept for treatment of cattle manure was studied. In batch experiments, a 10-24% increase of the specific methane yield from cattle manure and its fractions was obtained, when the substrates were inoculated with bacteria of the genus Caldicellusiruptor and Dictyoglomus. In a reactor experiment inoculation of a 68 degrees C pretreatment reactor with Caldicellusiruptor resulted in a 93% increase in the methane yield of the pretreatment reactor for a period of 18 days, but gave only a slight increase in the overall methane yield of the two-stage setup.     

Eat now,pay later? Evidence of deferred food-processing costs in diving seals

Seals may delay costly physiological processes (e.g. digestion) that are incompatible with the physiological adjustments to diving until after periods of active foraging. We present unusual profiles of metabolic rate (MR) in grey seals measured during long-term simulation of foraging trips (4-5 days) that provide evidence for this. We measured extremely high MRs (up to almost seven times the baseline levels) and high heart rates during extended surface intervals, where the seals were motionless at the surface. These occurred most often during the night and occurred frequently many hours after the end of feeding bouts. The duration and amount of oxygen consumed above baseline levels during these events was correlated with the amount of food eaten, confirming that these metabolic peaks were related to the processing of food eaten during foraging periods earlier in the day. We suggest that these periods of high MR represent a payback of costs deferred during foraging.     

Cloning,Expression, Purification,and Characterization of Biologically Active Recombinant Hemagglutinin-33, Type A Botulinum Neurotoxin Associated Protein

Botulinum neurotoxin type A, the most toxic substance known to mankind, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs) through polycistronic expression of a clustered group of genes. Hemagglutinin-33 (Hn-33) is a 33 kDa subcomponent of NAPs, which is resistant to protease digestion, a feature likely to be involved in the protection of the botulinum neurotoxin from proteolysis. In order to fully understand the function of Hn-33, large amounts of Hn-33 will be needed without dealing with biosafety risks to grow large cultures of C. botulinum. There are difficulties to clone the genes with the high A + T contents produced by C. botulinum. We report here for the first time using the Gateway technology to clone functional Hn-33 that has been expressed in E. coli. The yield of the recombinant Hn-33 was about 12 mg per liter of E. coli culture. The recombinant Hn-33 folds well in aqueous solution as shown with circular dichroism spectra, resists temperature-denaturation, is totally resistant to trypsin proteolysis despite the presence of cleavage sites on the molecular surface, and maintains its biological activities comparable to the native Hn-33 hemagglutination.     

Polyacrylamide lamination enables mass spectrometry compatible staining and in-gel digestion of proteins separated by agarose IEF

Agarose IEF enables the separation of large proteins and protein complexes. A complication of agarose gels attached onto polyester support is the lack of sensitive protein staining methods compatible with protein analysis and identification protocols. In this study, agarose IEF gels were used to separate the proteins, followed by layering the agarose with polyacrylamide. The formed laminate gels were seamless and durable and they were readily detached from the polyester. The gels were amenable to MS compatible staining. The sensitivity obtained with the acidic silver staining method was 20-50 ng/band of myoglobin. Laminated agarose was a suitable matrix for in-gel digestion based generation of tryptic peptides for MALDI-MS.     

Standardisation of rapid in-gel digestion by mass spectrometry

In-gel digestion has been standardised using a poly(propylene) disposable. We designed a four-step rapid and simple in-gel digestion protocol which is carried out in one self-contained reaction tube avoiding keratin contamination. In order to quantify the efficiency of in-gel digestion, we developed a rapid on-column peptide acetylation protocol. Results show that trypsin in-gel uptake is increased and in-gel digestion is 90% complete within 15 min. We further show that spectrum quality, peptide yield and sequence coverage for mass spectrometric analysis are enhanced. We utilise 2-D PAGE separation of photosystem II from barley to demonstrate that the protocol facilitates identification of highly hydrophobic membrane proteins.     

Adaptive neuro-fuzzy modelling of anaerobic digestion of primary sedimentation sludge

Modelling of anaerobic digestion systems is difficult because their performance is complex and varies significantly with influent characteristics and operational conditions. In this study, Adaptive Neuro-Fuzzy Inference System (ANFIS) were used for modelling of anaerobic digestion system of primary sludge of Kayseri municipal WasteWater Treatment Plant (WWTP). Effluent Volatile Solid (VS) and methane yield were predicted by the ANFIS. Two stage models were performed. In the first stage, effluent VS concentration was predicted using pH, VS concentration, flowrate of pre-thickened sludge and temperature of the influent as input parameters. In the second stage, effluent VS concentration in addition to first stage input parameters were used as input parameters to predict methane yield. The low Root Mean Square Error (RMSE) and high Index of agreement (IA) values were obtained with subtractive clustering method of a first order Sugeno type inference. The model performance was evaluated with statistical parameters. According to statistical evaluations, the models satisfactorily predict effluent VS concentration and methane yield.     

Organic waste conversion through anaerobic digestion: A critical insight into the metabolic pathways and microbial interactions

Anaerobic digestion is a promising method for energy recovery through conversion of organic waste to biogas and other industrial valuables. However, to tap the full potential of anaerobic digestion, deciphering the microbial metabolic pathway activities and their underlying bioenergetics is required. In addition, the behavior of organisms in consortia along with the analytical abilities to kinetically measure their metabolic interactions will allow rational optimization of the process. This review aims to explore the metabolic bottlenecks of the microbial communities adopting latest advances of profiling and ¹³C tracer-based analysis using state of the art analytical platforms (GC, GC-MS, LC-MS, NMR). The review summarizes the phases of anaerobic digestion, the role of microbial communities, key process parameters of significance, syntrophic microbial interactions and the bottlenecks that are critical for optimal bioenergetics and enhanced production of valuables. Considerations into the designing of efficient synthetic microbial communities as well as the latest advances in capturing their metabolic cross talk will be highlighted. The review further explores how the presence of additives and inhibiting factors affect the metabolic pathways. The critical insight into the reaction mechanism covered in this review may be helpful to optimize and upgrade the anaerobic digestion system.