Enhanced detection of autoantibodies on protein microarrays using a modified protein digestion technique

High-throughput studies to determine differential immune (humoral) response to diseases are becoming of increasing interest because the information they provide can help in early diagnosis as well as monitoring of therapeutics. Protein microarrays are a high-throughput and convenient technology that can be applied to the study of the humoral response. Proteins can be arrayed on slides and then probed with serum from different classes of patients to observe differences that may exist among autoantibodies that reflect differences in disease states. However, such studies may be difficult to interpret due to the weak overall signal response of such protein microarrays. We propose that this weak signal response is due to the physical positioning of the disease proteins that renders them sterically hindered from binding partners in the serum. In this study, we hypothesize that reducing the complexity and size of the disease proteins by chemical digestion using cyanogen bromide (CNBr) may enhance the overall signal from the humoral response and facilitate visualization of disease-specific responses in various classes of serum. A modified protein microarray methodology using CNBr digestion is presented here. The new workflow was applied to a set of 10 serum samples from healthy subjects, 10 from patients with chronic pancreatitis and 10 from patients diagnosed with pancreatic cancer and the results were compared to results obtained in the absence of CNBr digestion. CNBr digestion allowed the identification of 10 additional autoantibodies that responded to serum, 5 of which were unique to pancreatitis and cancer sera. This new methodology may increase the sensitivity of microarray studies measuring autoantibodies in serum.     

Effects of soybean oil and dietary copper levels on nutrient digestion,ruminal fermentation,enzyme activity,microflora and microbial protein synthesis in dairy bulls

ABSTRACT

The study evaluated the effects of soybean oil (SO) and dietary copper levels on nutrient digestion, ruminal fermentation, enzyme activity, microflora and microbial protein synthesis in dairy bulls. Eight Holstein rumen-cannulated bulls (14 ± 0.2 months of age and 326 ± 8.9 kg of body weight) were allocated into a replicated 4 × 4 Latin square design in a 2 × 2 factorial arrangement with factors being 0 or 40 g/kg dietary dry matter (DM) of SO and 0 or 7.68 mg/kg DM of Cu from copper sulphate (CS). The basal diet contained per kg DM 500 g of corn silage, 500 g of concentrate, 28 g of ether extract (EE) and 7.5 mg of Cu. The SO × CS interaction was significant (p < 0.05) for ruminal propionate proportion and acetate to propionate ratio. Dietary SO addition increased (p < 0.05) intake and total tract digestibility of EE but did not affect average daily gain (ADG) of bulls. Dietary CS addition did not affect nutrient intake but increased (p < 0.05) ADG and total tract digestibility of DM, organic matter, crude protein and neutral detergent fibre. Ruminal pH was not affected by treatments. Dietary SO addition did not affect ruminal total volatile fatty acids (VFA) concentration, decreased (p < 0.05) acetate proportion and ammonia N and increased (p < 0.05) propionate proportion. Dietary CS addition did not affect ammonia N, increased (p < 0.05) total VFA concentration and acetate proportion and decreased (p < 0.05) propionate proportion. Acetate to propionate ratio decreased (p < 0.05) with SO addition and increased (p < 0.05) with CS addition. Dietary SO addition decreased (p < 0.05) activity of carboxymethyl cellulase, cellobiase and xylanase as well as population of fungi, protozoa, methanogens, Ruminococcus albus and R. flavefaciens but increased (p < 0.05) α-amylase activity and population of Prevotella ruminicola and Ruminobacter amylophilus. Dietary CS addition increased (p < 0.05) activity of cellulolytic enzyme and protease as well as population of total bacteria, fungi, protozoa, methanogens, primary cellulolytic and proteolytic bacteria. Microbial protein synthesis was unchanged with SO addition but increased (p < 0.05) with CS addition. The results indicated that the addition of CS promoted nutrient digestion and ruminal fermentation by stimulating microbial growth and enzyme activity but did not relieve the negative effects of SO addition on ruminal fermentation in dairy bulls.     

Optimization of digestion parameters for protein quantification

We present a rapid and efficient in-solution enzymatic digestion protocol suitable for mass spectrometry-based absolute protein quantification techniques. The digestion method employs RapiGest SF (an acid-labile surfactant), an excess amount of modified trypsin (enzyme-to-substrate ratio of 2.5:1), and an incubation time of 2 h. No reduction/alkylation reagents are used. Digestion parameters were varied systematically to monitor their effect on rate and completeness of digestion. To demonstrate the general applicability of the method, the optimization was done using a viral hemagglutinin (HA) as a model protein and then applied to ricin, a potent protein toxin extracted from the castor bean (Ricinus communis). The parameters that were optimized included incubation time, concentration of RapiGest SF, enzyme-to-substrate ratio, and incubation temperature. The optimization was done by comparing the yields from two protein-specific peptides originating from two different sites of the HA protein. The analysis was performed by liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode using isotopically labeled peptide standards for quantification.     

In vitro protein digestion kinetics of protein sources for pigs

In current feed evaluation systems, the nutritional value of protein sources in diets for pigs is based on the ileal digestibility of protein and amino acids, which does not account for the kinetics of protein digestion along the gastrointestinal tract. The objective of the present study was to determine the in vitro protein digestion kinetics of different protein sources (soya bean meal (SBM), wheat gluten (WG), rapeseed meal (RSM), whey powder (WP), dried porcine plasma protein, yellow meal worm larvae and black soldier fly larvae (BSF)). Protein sources were incubated with pepsin at pH 3.5 for 0 to 90 min and subsequently with pancreatin at pH 6.8 for 0 to 210 min at 39°C. The in vitro protein digestion kinetics were described as the kinetics of nitrogen (N) solubilisation and the release of low molecular weight peptides (LMW) (<500 Da). The N solubilisation rate ranged from 0.025 min⁻¹ for BSF to 0.685 min⁻¹ for WP during the incubation with pepsin, and from 0.027 min⁻¹ for RSM to 0.343 min⁻¹ for WP during the incubation with pancreatin. The release rate of LMW peptides ranged from 0.027 min⁻¹ for WG to 0.093 min⁻¹ for WP during the incubation with pepsin, and from 0.029 min⁻¹ for SBM to 0.385 min⁻¹ for WP. Black soldier fly larvae showed a similar release rate of LMW peptides as WP during the incubation with pancreatin. At the end of the sequential incubation with pepsin (90 min) and pancreatin (210 min), WG and WP showed the highest percentage of N present in LMW peptides relative to total N (78% and 79%, respectively), whereas SBM showed the lowest (35%). In conclusion, protein sources for pig diets show substantial differences in in vitro protein digestion kinetics as measured by the kinetics of N solubilisation and the release of LMW peptides. The rate of release of LMW peptides was not correlated to the rate of N solubilisation for each of the protein sources evaluated.     

Negative chromatography on agarose-TREN as a technique for purification of protein spiked in soybean seeds extract

Alkyl amines and polyamines have been used as ligands for protein purification by mixed-mode chromatography. The adsorption of proteins onto these ligands seems to be governed by multiple effects such as electrostatic, hydrophobic, and affinity interactions. In this work we investigated the adsorption of proteins extracted from soybean onto the adsorbent agarose-Tris(2-aminoethyl)amine (TREN). The effects of flow rate, buffer system, and extract concentration on the capture of proteins extracted from soybean were evaluated. Experiments using Mes at pH 6.5 as adsorption buffer allowed the adsorption of almost the totality of native soybean protein with a dynamic adsorption capacity of 13.50 mg mL^?1 adsorbent. Experiments with human IgG (pI in the range of 5.8–9.0) and human serum albumin (HSA, pI of 4.9) spiked into these extracts lead to the conclusion that electrostatic forces play a major role in the interaction between protein and agarose-TREN. Based on this work, negative chromatography with agarose-TREN should be considered as a method for purification of basic recombinant protein produced in transgenic soybean seeds.     

Fiber-packed channel bioreactor for microfluidic protein digestion

The fabrication and performance of a fiber-packed channel bioreactor in microchip along with its application in protein analysis were reported. The feasibility and performance of the unique microfluidic bioreactor were demonstrated by the tryptic digestion of myoglobin (MYO) and BSA. The on-chip digestion was carried out at a flow rate of 2.0 microL/min and the digestion time was significantly reduced to less than 5 s. The digests were identified by MALDI-TOF MS with sequence coverages of 66% (MYO) and 40% (BSA) that were comparable to those obtained by the conventional in-solution tryptic digestion. The present fiber-based microchip bioreactor provides a promising platform for the high-throughput protein identification.     

Effects of non-enzymatic browning reaction intensity on in vitro ruminal protein degradation and intestinal protein digestion of soybean and cottonseed meals

The effects of non-enzymatic browning reactions on in vitro ruminal gas production and in vitro ruminal and intestinal crude protein (CP) digestibilities of soybean (SBM) and cottonseed (CSM) meals were investigated. Non-enzymatically browned SBM and CSM samples were prepared using two xylose levels (10 or 30 g/kg dry matter), two heating lengths (30 or 60 min) and two heating temperatures (120 or 150 °C) for a total of one untreated (commercially solvent-extracted, Control) and eight treated samples for each protein source. The control SBM had higher (P<0.001) in vitro ruminal CP degradability values than the treated samples. Intestinal protein digestibility and total-tract CP digestibility of CSM and SBM were affected by the treatment (P<0.01). The results of the study indicate that not only ruminal CP degradability is reduced but also intestinal and total-tract CP digestibilities may be lowered depending on protein source and intensity of the non-enzymatic browning reaction.     

Crystallographic data for soybean hydrophobic protein

The soybean hydrophobic protein belongs to a family of proteins that contains a number of storage and phospholipid binding proteins. Its function is not known, but its overall hydrophobic nature is typical of many membrane proteins of similar size. The molecular weight is 8.3 x 10(3), and it crystallizes in the space group P2(1)2(1)2(1), with a = 52.01 A, b = 43.50 A and c = 28.80 A. The crystals diffract to 1.8 A resolution, and are thus suitable for X-ray structural studies.     

Carbon balances for in vitro digestion and fermentation of potential roughages for pregnant sows

Ad libitum feeding of pregnant sows requires satiating, intake-restricting feed components to prevent sows from getting excessively fat. Because hindgut fermentation starts only after and proceeds much slower than enzymatic digestion in the small intestine, fermentation products might, as nutrients, induce a prolonged physiological satiation of sows. To simulate hindgut fermentation and determine the fermentative release of short-chain fatty acids (SCFA) in vitro, gas production tests (GPTs) were performed with different raw materials after removal of enzymatically hydrolysable compounds. Fresh feces from sows that received standard sow feed was used as inoculum. Fresh, ensiled, or dehydrated by-products of the food industry (brewers’ grains, liquid yeast feed, maize gluten feed, raw potato chips, potato steam peel, pressed potato pulp, sugar beet pulp (SBP)) and whole-plant products (grass, maize) were tested as individual products.

Balances were drawn up of the in vitro flow of organic carbon (OC) to an “ileal” nutrient fraction, a “hindgut” fraction of SCFA and gas, and “fecal” remnants. The OC balances revealed large variations among raw materials in terms of their contribution to the different fractions. Potato steam peel gave the largest “ileal” nutrient fraction (77% of total OC), the lowest was observed with fresh sugar beet pulp (9% of total OC). In the GPT, SBP, and potato pulp brought about the highest “hindgut” SCFA yields (32–49% of total OC), and together with raw potato chips, the highest amounts of gas. Grass products and liquid yeast feed were slower fermented than most by-products. Moreover, whole-plant materials gave larger “fecal” OC portions than by-products, with the exception of fresh and dehydrated brewer’s grains. Together with straw, the latter were the least degradable of all raw materials tested. Among the grass products, dehydrated grass, and among the by-products, raw potato chips left the least “fecal” residues, i.e. they were nutritionally utilized to the largest extent.     

An on-line technique for monitoring propionic acid fermentation

An on-line technique, based on measuring the increase in pressure due to CO₂ release in a closed air-tight reactor, was used to evaluate the fermentation of lactate by propionibacteria. The method was applied to batch cultures of Propionibacterium shermanii grown in yeast extract/sodium lactate medium containing lactate as a carbon source under micro-aerophilic conditions. Gas pressure evolution was compared both with substrate consumption and metabolites production and with acidification and growth. Linear relationships were found between gas pressure variation, lactate consumption and propionate and acetate production. The technique also enabled the evaluation of total CO₂ produced, by taking account of pressure, oxygen and pH measurements. These results tend to show that this simple and rapid method could be useful to monitor propionic acid bacteria growth.     

Microwave-assisted specific chemical digestion for rapid protein identification

We have developed a rapid microwave-assisted protein digestion technique based on classic acid hydrolysis reaction with 2% formic acid solution. In this mild chemical environment, proteins are hydrolyzed to peptides, which can be directly analyzed by MALDI-MS or ESI-MS without prior sample purification. Dilute formic acid cleaves proteins specifically at the C-terminal of aspartyl (Asp) residues within 10 min of exposure to microwave irradiation. By adjusting the irradiation time, we found that the extent of protein fragmentation can be controlled, as shown by the single fragmentation of myoglobin at the C-terminal of any of the Asp residues. The efficacy and simplicity of this technique for protein identification are demonstrated by the peptide mass maps of in-gel digested myoglobin and BSA, as well as proteins isolated from Escherichia coli K12 cells.     

In vitro digestion and fermentation characteristics of canola co-products simulate their digestion in the pig intestine

Canola co-products are sources of amino acid and energy in pig feeds, but their fermentation characteristics in the pig intestine are unknown. Thus, we determined the in vitro fermentation characteristics of the canola co-products Brassica juncea solvent-extracted canola meal (JSECM), Brassica napus solvent-extracted canola meal (NSECM), B. napus expeller-pressed canola meal (NEPCM) and B. napus cold-pressed canola cake (NCPCC) in comparison with soybean meal (SBM). Samples were hydrolysed in two steps using pepsin and pancreatin. Subsequently, residues were incubated in a buffer solution with fresh pig faeces as inocula for 72 h to measure gas production. Concentration of volatile fatty acids (VFA) per gram of dry matter (DM) of feedstuff was measured in fermented solutions. Apparent ileal digestibility (AID) and apparent hindgut fermentation (AHF) of gross energy (GE) for feedstuffs were obtained from pigs fed the same feedstuffs. On DM basis, SBM, JSECM, NSECM, NEPCM and NCPCC contained 15, 19, 22, 117 and 231 g/kg ether extract; and 85, 223, 306, 208 and 176 g/kg NDF, respectively. In vitro digestibility of DM (IVDDM) of SBM (82.3%) was greater (P<0.05) than that of JSECM (68.5%), NSECM (63.4%), NEPCM (67.5%) or NCPCC (69.8%). The JSECM had greater (P<0.05) IVDDM than NSECM. The IVDDM for NSECM was lower (P<0.05) than that for NEPCM, which was lower (P<0.05) than that for NCPCC. Similarly, AID of GE was greatest for SBM followed by NCPCC, JSECM, NEPCM and then NSECM. Total VFA production for SBM (0.73 mmol/g) was lower (P<0.05) than that of JSECM (1.38 mmol/g) or NSECM (1.05 mmol/g), but not different from that of NEPCM (0.80 mmol/g) and NCPCC (0.62 mmol/g). Total VFA production of JSECM was greater (P<0.05) than that of NSECM. Total VFA production of NSECM was greater (P<0.05) than that of NEPCM or NCPCC, which differed (P<0.05). The ranking of feedstuffs for total VFA production was similar to AHF of GE. In conclusion, in vitro fermentation characteristics of canola co-products and SBM simulated their fermentation in the small and large intestine of pigs, respectively. The 30% greater VFA production for JSECM than NSECM due to lower lignified fibre of JSECM indicates that fermentation characteristics differ between canola species. The NSECM had the highest fermentability followed by NEPCM and then NCPCC, indicating that fat in canola co-products can limit their fermentability in the hindgut.     

Feed digestion,rumen fermentation and blood biochemical constituents in Malpura rams fed a complete feed-block diet with the inclusion of tree leaves

This experiment assayed the influence of the inclusion of dried Azardirachta indica, Albizzia lebbek or Ailanthus excelsa leaves in pearl millet stover-based complete feed block diets on feed intake, nutrient utilization, rumen fermentation characteristics, ciliate protozoa population and blood biochemical constituents in adult Malpura sheep. Complete feed blocks were formulated to have roughage-to-concentrate ratio of 70:30. Pearl millet stover (PMS) was used as basal roughage; 30 parts of pearl millet stover was replaced with dried leaves either of Azardirachta indica (NL), Albizzia lebbek (SL) or Ailanthus excelsa (AL). Twelve hogget Malpura rams, divided into four equal groups, were offered one of the four dietary treatments. A feeding-cum-metabolic trial was conducted to assess nutrient utilization. Rumen liquor samples were collected at 0, 3, 6, 12, 18 and 24 h post-feeding to assess rumen fermentation pattern and ciliate protozoa population. Inclusion of dried leaves in PMS-based diets improved CP and DCP content. Dietary DCP was low (P < 0.01) in PMS (8.52%) compared to tree leaves (9.77–11.59%) diets. AL and NL diets had higher (P < 0.05) DCP than the SL diet. The inclusion of tree leaves did not influence organic matter, crude protein or cellulose digestibility, but depressed dry matter, NDF, ADF and energy digestibility. DE content was also lower in tree leave diets. Inclusion of tree leaves improved CP and DCP intake, but DE intake and nitrogen utilization did not change. The pH of rumen liquor (SRL) was low (6.99, P < 0.05), but total nitrogen (52.9 mg/dl SRL) and NH₃-nitrogen (9.34 mg/dl SRL) concentrations were higher (P < 0.01) in the AL diet. TVFA concentrations and ciliate protozoa population were similar on the four diets. Animals in the four groups had the desired concentration of rumen metabolites required for fibrous diets. Complete feed-block feeding provided a constant nutrient supply to rumen microbes that optimise rumen fermentation. Blood biochemical constituents did not change due to the inclusion of tree leaves. Therefore, tree leaves can be included with roughage-based feeding to improve the protein nutrition status of ruminants. Further studies are required to assess the negative influence of tree leaves on digestibility.     

Sauerkraut fermentation : New fermentation vat

Summary A new sauerkraut fermentation vat with external recycling of brine is described. This system allows the control of fermentation, improves the homogeneity of final product and accelerates theacidification by the usual lactic-acid bacteria.     

Comparison of protein fermentation characteristics in rumen fluid determined with the gas production technique and the nylon bag technique

In this study, a modified version of the gas production technique was used to determine protein fermentation characteristics in rumen fluid of 19 feedstuffs. Performing the incubations in a N-free environment, and with an excess of rapidly fermentable carbohydrates, made N the limiting factor to microbial growth, and so gas production profiles reflected the availability of N from the feed samples. Results showed that fermentation of protein in rumen fluid can be determined with this modified gas production technique, and that there were distinct differences in protein fermentation between the feed samples. Availability of protein for fermentation was highest in wheat, potato pieces and lupin, and lowest in Rumiraap, a formaldehyde treated rapeseed meal, palm kernel expeller and brewery grains. The protein degradation characteristics of the 19 feed ingredients were also determined with the in situ nylon bag technique. With the obtained results, the amount of rumen escape protein (REP) was calculated for each feedstuff. The results showed that the rate of degradation ranged from 0.010/h for Rumiraap to 0.151/h for wheat. The amount of REP ranged from 197 g/kg CP for lupin to 840 g/kg CP for Rumiraap. Comparing the gas production results with the results obtained with the nylon bag technique showed that there was a good relationship between the gas production after 12–25 h of incubation and the calculated amount of REP (r² = 0.83–0.85). The results show that the adapted gas production technique, being depleted of N and using an excess of rapidly fermentable carbohydrates, is suitable to recognize differences in N availability between feed samples and can be used as an alternative to the nylon bag technique and other in vitro techniques.     

液态发酵豆粕制备纳豆激酶方法的优化

纳豆激酶是一种丝氨酸蛋白酶,具有很强的纤溶活性,由于具有安全性好、作用迅速持久、成本低等优点,适合用于开发新一代的溶栓剂或保健食品,具有广阔的市场前景。本研究探讨了以豆粕为原料液态发酵豆粕生产纳豆激酶的发酵方法。首先通过单因素实验发现影响产酶的主要因素有接菌量、发酵时间、培养基pH及豆粕含量,再由正交实验得到最优组合为接菌量1%,豆粕含量2%,pH为7.0,发酵时间48h,该条件下发酵酶活力最高达到4 429.6U/mL。本研究确定了以豆粕为原料制备纳豆激酶的最佳条件,为豆粕的合理使用和纳豆激酶的工业化生产提供了实验依据。     

Solid-substrate fermentation of alfalfa for enhanced protein recovery

Solid-substrate fermentations for extraction of protein from pressed alfalfa residues with Aspergillus sp. QM 9994 aspergillus niger QM 877, and Rhizopus nigricans QM 387 were conducted in shake flasks. Upon reimbibing and second pressing, total protein recovery from alfalfa was increased from 47.2% for control samples and up to 64.5% for fermented samples. Analysis of juice from fermented samples indicated the presence of cellulase as well as pectinase activities. Dialysis cultures of cellulase-producing fungi showed that total biomass production and solids consumption were much higher than those of a mutant strain lacking the ability to produce cellulase, indicating significant utilization of cellulosic materials in alfalfa. The biomass yields in the former case ranged from 39–47% based on total solids consumption. Since some of the cellulosic and other carbohydrate constituents in alfalfa may be converted into fungal protein, final alfalfa residues following protein extraction in a commercial process would be less bulky for storage and handing and would be more digestible as a nonruminant animal feed.     

Association of bacteria with the fungal fermentation of soybean tempe

Bacteria grew to viable populations of 10⁸–10⁹ cfu/g during the fermentation of soybeans into tempe with the fungus, Rhizopus oligosporus. Bacillus pumilus and B. brevis were the predominant bacterial species, reaching populations of approximately 10⁸ cfu/g during the 48 h fermentation. Species of Streptococcus faecium, Lactobacillus casei, Klebsiella pneumoniae and Enterobacter cloacae also contributed to the fermentation and achieved populations of 10⁶–10⁷ cfu/g. and accepted 25 May 1989