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PubMed2002

On-probe digestion of bacterial proteins for MALDI-MS.

On-probe digestion combined with MALDI mass spectrometry is studied as a rapid method for the analysis and identification of bacterial proteins. The use of trypsin adsorbed to the probe surface reduces the digestion time from hours to minutes. A high amount of trypsin must be applied to the probe for the successful digestion of bacterial proteins. Mass spectra of the digest contain a number of low-mass digest fragments. Several components of a B. subtilis bacterial digest can be identified through postsource decay and database searching.

Bacillus subtilis↗
PubMed2004

Enzymatic tissue digestion as an alternative sample preparation approach for quantitative analysis using liquid chromatography-tandem mass spectrometry.

Compound extraction from biological tissue often presents a challenge for the bioanalytical chemist. Labor-intensive homogenization or sonication of whole or powdered tissue is performed before compounds can be extracted and analyzed. Enzymatic digestion is commonly used for tissue dissociation and cell harvesting and offers the advantages of unattended sample preparation, potential automation, and low cost. The feasibility of enzymatic digestion as an alternate tissue preparation technique was evaluated for bioanalysis of drugs in conjunction with LC/MS/MS. Two different enzymes (collagenase and proteinase K) that are known to degrade connective tissues to allow tissue dissolution were chosen for evaluation, employing well-known antidepressants desipramine and fluoxetine as test compounds in dog and rat brain tissue. Comparison between enzymatic digestion and conventional homogenization tissue preparation was performed, including investigation of matrix ionization suppression of both methods using a postcolumn infusion system. Results showed that enzymatic digestion has extraction efficiency comparable to homogenization. Matrix ionization suppression was not observed for either the test compounds evaluated or the sample extraction method. Test compound levels of incurred tissue samples prepared by enzymatic digestion were in good agreement with the values obtained by the conventional homogenization tissue preparation, indicating that enzymatic digestion is an appropriate tissue sample preparation method.

Animals↗
PubMed1993

Tryptic digestion of the human erythrocyte glucose transporter: effects on ligand binding and tryptophan fluorescence.

The conformation of the human erythrocyte glucose transport protein has been shown to determine its susceptibility to enzymatic cleavage on a large cytoplasmic loop. We took the converse approach and investigated the effects of tryptic digestion on the conformational structure of this protein. Exhaustive tryptic digestion of protein-depleted erythrocyte ghosts decreased the affinity of the residual transporter for cytochalasin B by 3-fold but did not affect the total number of binding sites. Tryptic digestion also increased the affinity of the residual transporter for D-glucose and inward-binding sugar phenyl beta-D-glucopyranoside but decreased that for the outward-binding 4,6-O-ethylidene glucose. These results suggest that tryptic cleavage stabilized the remaining transporter in an inward-facing conformation, but one with decreased affinity for cytochalasin B. The steady-state fluorescence emission scan of the purified reconstituted glucose transport protein was unaffected by tryptic digestion. Addition of increasing concentrations of potassium iodide resulted in linear Stern-Volmer plots, which were also unaffected by prior tryptic digestion. The tryptophan oxidant N-bromosuccinimide was investigated to provide a more sensitive measure of tryptophan environment. This agent irreversibly inhibited 3-O-methylglucose transport in intact erythrocytes and cytochalasin B binding in protein-depleted ghosts, with a half-maximal effect observed for each activity at about 0.3-0.4 nM. Treatment of purified glucose transport protein with N-bromosuccinimide resulted in a time-dependent quench of tryptophan fluorescence, which was resolved into two components by nonlinear regression using global analysis. Tryptic digestion retarded the rate of oxidation of the more slowly reacting class of tryptophans. (ABSTRACT TRUNCATED AT 250 WORDS)

3-O-Methylglucose↗
PubMed1983

Periodicity of exonuclease III digestion of chromatin and the pitch of deoxyribonucleic acid on the nucleosome.

Exonuclease III has previously been shown to pause about every 10 nucleotides along the 3' strands while it invades the nucleosome core. Here, the exact periodicity of this digestion, i.e., the spacing of the pauses, was determined. Results showed that the exonuclease digests the first 20 nucleotides at the edge of the nucleosome core with a periodicity of approximately 11 nucleotides; in contrast, DNA closer to the center of the particle is digested with a smaller periodicity of about 10 nucleotides. These figures differ from the known periodicity of DNase I digestion, approximately 10 and 10.5 nucleotides at the edge and in the center of the nucleosome, respectively. Moreover, as shown by sedimentations in sucrose gradients, the structure of the nucleosome does not appear to be significantly altered by the gradual destruction of its DNA moiety by the exonuclease. Such stability of the nucleosome, along with other complementary observations, indicates that the transition in the digestion periodicity of the exonuclease may not be the consequence of a structural rearrangement of the particle upon trimming. This transition may rather be ascribed to the properties of the native nucleosome and to the intrinsic mechanism of action of the enzyme. Finally, evidence is presented which suggests that the exonuclease 10-nucleotide periodicity of digestion of the inner region of the nucleosome reflects a 10 base pair/turn pitch of the DNA in that region.

Animals↗
PubMed1984

Tryptic digestion of rabbit skeletal myofibrils: an enzymatic probe of myosin cross-bridges.

Tryptic digestion of rabbit skeletal myofibrils under physiological ionic strength and pH conditions was used as a probe of cross-bridge interaction with actin in the presence of nucleotides and pyrophosphate. Under rigor conditions, digestion of myofibrils at 24 degrees C results in the formation of 25K, 110K [heavy meromyosin (HMM)], and light meromyosin (LMM) fragments as the main reaction products. Very little if any 50K peptide is generated in such digestions. In the presence of magnesium pyrophosphate, magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and MgATP, the main cleavage proceeds at two positions, 25K and 75K from the N-terminal portion of myosin, yielding the 25K, 50K, and 150K species. The relative amounts of the 50K, 110K, and 150K peptides and the rates of myosin heavy-chain digestion in the presence of pyrophosphate and AMPPNP indicate partial dissociation of myosin from actin. Direct centrifugation measurements of the binding of HMM and subfragment 1 (S-1) to actin in myofibrils confirm that cross-bridges partition between attached and detached states in the presence of these ligands. In the presence of MgADP, HMM and S-1 remain attached to actin at 24 degrees C. However, tryptic digestion of myofibrils containing MgADP is consistent with the existence of a mixed population of attached and detached cross-bridges, suggesting that only one head on each myosin molecule is attached to actin. As shown by tryptic digestion of myofibrils and the measurements of HMM and S-1 binding to actin, nucleotide- and pyrophosphate-induced dissociation of cross-bridges is more pronounced at 4 than at 24 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Trypsin digestion of junctional sarcoplasmic reticulum vesicles.

A putative constituent of the junctional processes, connecting the terminal cisternae of sarcoplasmic reticulum and the transverse tubules of skeletal muscle fibers, is a greater than or equal to 350,000-dalton (Da) protein that displays ryanodine binding and Ca2+ channel properties. Ryanodine modulation of Ca2+ fluxes suggests that the ryanodine receptor and calcium channel are integral parts of one functional unit corresponding to the greater than or equal to 350,000-Da protein [Inui, M., Saito, E., & Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747; Campbell, K. P., Knudson, C. M., Imagawa, T., Leung, A. L., Sutko, J. L., Kahl, S. D., Raab, C. R., & Madson, L. (1987) J. Biol. Chem. 262, 6460-6463]. We subjected vesicular fragments of junctional-cisternal membrane to stepwise trypsin digestion. The greater than or equal to 350,000-Da protein is selectively cleaved in the early stage of digestion, with consequent disappearance of the corresponding band in electrophoretic gels. The Ca2+-ATPase is cleaved at a later stage, while calsequestrin is not digested under the same experimental conditions. While the Ca2+-ATPase yields two complementary fragments that are relatively resistant to further digestion, the greater than or equal to 350,000-Da protein yields fragments that are rapidly broken down to small peptides. Under conditions producing extensive digestion of the greater than or equal to 350,000-Da protein, the junctional processes are still visualized by electron microscopy, with no discernible alterations of their ultrastructure. The functional properties of the Ca2+ release channel are also maintained following trypsin digestion, including blockage by Mg2+ and ruthenium red and activation by Ca2+ and nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Isolation and enzymatic digestion of body complex of soybean seed.

The body complex of the soybean seed (BCSS) was isolated from the single cells (27.2%) by a sequential procedure of autoclaving with water, cellulase digestion for the primary cell wall, pectinase digestion for the secondary cell wall, and defatting with hexane washing. Its characteristics were then investigated. The defatted BCSS (DBCSS) consisted of protein (76.5%) and mannose-rich carbohydrates (3.2%). Screening of the food-processing protease for the digestion of DBCSS was carried out, and a kind of alkaline protease was selected. The inner protein of DBCSS was easily extracted with 0.1 M sodium carbonate buffer, pH 10, and the insoluble shell of the body complex (SDBCSS) was left. SDBCSS consisted of hydrophobic amino acid-rich protein. SDBCSS was easily digested by the selected alkaline protease. SDBCSS was dissolved by boiling with sodium dodecyl sulfate-mercaptoethanol, and it was found to consist of a protein of approximately 3 kDa. The high enzymatic digestion including the selected protease for soybean seed and defatted soybean meal was carried out; both were extracted and digested with a yield of >99.5%. The final indigestible residue was found as paired hexagonal and filamentous organs of the soybean cells.

Amino Acids↗
PubMedDate not supplied

Microcolumn capture and digestion of proteins combined with mass spectrometry for protein identification.

A procedure has been developed for protein identification using mass spectrometry (MS) that incorporates sample cleanup, preconcentration, and protein digestion in a single-stage system. The procedure involves the adsorption of a protein, or protein mixture, from solution onto a hydrophobic resin that is contained within a microcolumn. Sample loading is accomplished by flowing the protein solution through the microcolumn, where the protein adsorbs to the hydrophobic surface. The protein is digested while still bound to the hydrophobic surface by flowing a buffered trypsin solution through the column bed. The peptide fragments are subsequently eluted for detection by MALDI or ESI-MS. The procedure is demonstrated using dilute protein samples containing high concentrations of salt, urea, and modest amount of sodium dodecyl sulfate relative to protein. Peptide fragments are also detected by MS from a 500 nM bacteriorhodopsin solution digested in a microcolumn. In this case, a combined cyanogen bromide/trypsin digestion was performed in-column. The procedure is applied to the MALDI-MS/MS identification of proteins present in an individual fraction collected by ion exchange HPLC separation of E. coli total cell extract. An additional application is illustrated in the analysis of a human plasma fraction. A total of 14 proteins, which were present in the sample at sub-micromolar concentrations, were identified from ESI-MS/MS. The microcolumn digestion procedure represents the next step toward a system for fully automated protein analysis through capture and digestion of the adsorbed protein on hydrophobic surfaces.

Chromatography, Ion Exchange↗
PubMed2006

A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea.

Proteolytic digestion of a complicated protein mixture from an organelle or whole-cell lysate is usually carried out in a dilute solution of a denaturing buffer, such as 1-2 M urea. Urea must be subsequently removed by C18 beads before downstream analysis such as HPLC/MS/MS or complete methylation followed by IMAC isolation of phosphopeptides. Here we describe a procedure for digesting a complicated protein mixture in the absence of denaturants. Proteins in the mixture are precipitated with trichloroacetic acid/acetone for denaturation and salt removal and resuspended in NH4HCO3 buffer. After trypsinolysis, the resulting peptides are not contaminated by urea or other nonvolatile salts and can be dried in a SpeedVac to remove NH4HCO3. When this protocol was applied to an extract of A431 cells, 96.8% of the tryptic peptides were completely digested (i.e., had no missed cleavage sites), in contrast to 87.3% of those produced by digestion in urea buffer. We successfully applied this digestion method to analysis of the phosphoproteome of adiposomes from HeLa cells, identifying 33 phosphorylation sites in 28 different proteins. Our digestion method avoids the need to remove urea before HPLC/MS/MS analysis or methylation and IMAC, increasing throughput while reducing sample loss and contamination from sample handling. We believe that this method should be valuable for proteomics studies.

Amino Acid Sequence↗
PubMedDate not supplied

Hyaluronan digestion and synthesis in an experimental model of metastatic tumour.

To approach the question of hyaluronan catabolism in tumours, we have selected the cancer cell line H460M, a highly metastatic cell line in the nude mouse. H460M cells release hyaluronidase in culture media at a high rate of 57 pU/cell/h, without producing hyaluronan. Hyaluronidase was measured in the H460M cell culture medium at the optimum pH 3.8, and was not found above pH 4.5, with the enzyme-linked sorbent assay technique and zymography. Tritiated hyaluronan was digested at pH 3.8 by cells or cell membranes as shown by gel permeation chromatography, but no activity was recorded at pH 7 with this technique. Hyaluronan was digested in culture medium by tumour slices, prepared from tumours developed in nude mice grafted with H460M cells, showing that hyaluronan could be digested in complex tissue at physiological pH. Culture of tumour slices with tritiated acetate resulted in the accumulation within 2 days of radioactive macromolecules in the culture medium. The radioactive macromolecular material was mostly digested by Streptomyces hyaluronidase, showing that hyaluronan was its main component and that hyaluronan synthesis occurred together with its digestion. These results demonstrate that the membrane-associated hyaluronidase of H460M cells can act in vivo, and that hyaluronan, which is synthesised by the tumour stroma, can be made soluble and reduced to a smaller size by tumour cells before being internalised and further digested.

Animals↗
PubMed2002

The effects of displacement rate and proteoglycan digestion on the fracture resistance of tissue grown from chondrocyte culture.

Fracture toughness of cartilage and cartilage replacement tissues is important in injury and disease. For example, cartilage is thought to weaken before it fibrillates in the disease osteoarthritis. Since both loading rate and proteoglycan content affect viscoelastic properties, they may both affect fracture toughness of cartilage and cartilage analogs. In this study, fracture toughness of tissue grown in chondrocyte culture was measured as a function of loading rate and proteoglycan digestion. Control tissue and tissue digested with chondroitinase ABC (cABC) to remove proteoglycans were tested at displacement rates of 0.1 and 0.5 mm/sec. Displacement rate had no effect on fracture toughness for either control or digested tissue. Proteoglycan digestion reduced tissue thickness by 30% and when evaluated on a material basis increased fracture toughness. There was no interaction between digestion and loading rate. When the fracture toughness was normalized to collagen content, which removed the effect of tissue shrinkage, there was no effect of proteoglycan digestion on fracture toughness. These data suggest that proteoglycans do not contribute to tissue toughness, other than by reducing thickness and increasing collagen density.

Journal Article↗
PubMed1997

Automated protein preparation techniques using a digest robot.

Since the introduction of fast analysis methods for peptide mixtures such as MALDI-MS, peptide micropreparation and digest methods have become an important bottleneck in the protein characterization process. We therefore developed and describe here a digest robot capable of processing 30 protein samples in parallel [Houthaeve et al. (1995), FEBS Lett. 376, 91-94]. Briefly, after gel pieces or blots are cut out, they are loaded in flowthrough reactors and these are loaded in a thermocontrolled reactor block. The proteins are then washed, reduced, and alkylated, proteolytically or chemically cleaved, and resulting peptides extracted. The system allows the parallel use of different reagents and enzymes during the same run, and is compatible with RP-HPLC peptide separation and Edman degradation, MALDI-MS, and NanoES-MS/MS. The digest robot is now also commercially available from ABIMED. In an ongoing project aimed at elucidating proteinaceous structures involved in the functional and structural maintenance of the Golgi apparatus, we illustrate the strength of the digest robot for the fast analysis of several proteins. We conclude that the performance of the digest robot is comparable to currently used manual digestion methods. The approach outlined makes sample preparation procedures faster, simpler, and less labor-intensive.

Chemistry Techniques, Analytical↗
PubMed2001

Rice alpha-mannosidase digesting the high mannose glycopeptide of glutelin.

alpha-Mannosidase (EC 3.2.1.24) from rice dry seeds was purified to homogeneity. Optimum pH and Km for pNP-alpha-Man hydrolysis were pH 4.3-4.5 and 1.04 mM, respectively. The enzyme digested mannobioses such as Manalpha-1,2Man, Manalpha-1,6Man, Manalpha-1,3Man but Manalpha-1,4Man. Zn2+ ion was required for the activity, whereas EDTA and swainsonine inhibited the activity by 80 and 96%, respectively. The rice storage protein, glutelin was prepared and its basic subunits were shown to have high mannose-type sugar chains by two-dimensional mapping using NH2-P and C18 silica columns. They were Man9GlcNAc2, Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2 and Man5GlcNAc2. All these oligosaccharides were digested by the purified alpha-mannosidase, and Man-GlcNAc2 and mannose were formed. Glycopeptides, having these high mannose-type sugar chains, could also be digested by the alpha-mannosidase. Subunits were prepared from glutelin basic subunit and the richest subunit among them, subunit 2 (isoform 2), was digested by the alpha-mannosidase. Isoform 2 was digested by V8 protease only partially and slowly. However, isoform 2, pre-treated with the alpha-mannosidase, was rapidly and completely digested by V8 protease.

Journal Article↗
PubMed1995

Investigation of decomposition products of microwave digestion of food samples.

The involatile residues remaining after closed-vessel microwave digestion of various food samples, using nitric acid, with and without post-digestion treatment with hydrogen peroxide, have been studied. Decomposition products were found to include aliphatic and aromatic acids, nitro-compounds, oxalates and inorganic nitrates and phosphates. Measures of digestion completeness were provided by appearance, carbon content, infrared spectra and thin layer chromatograms of the residues, enabling a comparison of different digestion methods and sample types. Residual carbon levels varied linearly with the relative amounts of carboxylic acid and inorganic nitrate, as measured by infrared spectrometry of the residues. The formation of calcium oxalate was also a function of the degree of decomposition: the carboxylic acid: oxalate ratio increased in a logarithmic fashion with increasing residual carbon content. Particularly high levels of residual carbon from nitric acid digestion of milk powder, due largely to carboxylic acid residues, were substantially decreased by post-digestion treatment with hydrogen peroxide. However, nitrobenzoic acids, which proved major interferents in electrochemical analysis, were only removed by treatment with perchloric acid.

Food↗
PubMed2003

A microchip-based proteolytic digestion system driven by electroosmotic pumping.

Microchip-based proteomic analysis requires proteolytic digestion of proteins in microdevices. Enzyme reactors in microdevices, fabricated in glass, silicon, and PDMS substrates, have recently been demonstrated for model protein digestions. The common approach used for these enzyme reactors is employment of a syringe pump(s) to generate hydrodynamic flow, driving the proteins through the reactors. Here we present a novel approach, using electroosmotic flow (EOF) to electrokinetically pump proteins through a proteolytic system. The existence of EOF in the proteolytic system packed with immobilized trypsin gel beads was proven by imaging the movement of a neutral fluorescent marker. Digestions of proteins were subsequently carried out for 12 min, and the tryptic peptides were analyzed independently using capillary electrophoresis (CE) and MALDI-TOF mass spectrometry (MS). The results from CE analysis of the tryptic peptides from the EOF-driven proteolytic system and a conventional water bath digestion were comparable. MALDI-TOF MS was used to identify the parent protein and the tryptic peptides using MS-Fit database searching. The potential utility of the EOF-driven proteolytic system was demonstrated by direct electro-elution of proteins from an acrylamide gel into the proteolytic system, with elution and tryptic digestion achieved in a single step. The EOF-driven proteolytic system, thus, provides a simple way to integrate protein digestion into an electrophoretic micro total analysis system for protein analysis and characterization.

Electrochemistry↗
PubMed1999

Effect of process temperature, pH and suspended solids content upon pasteurization of a model agricultural waste during thermophilic aerobic digestion.

Thermophilic aerobic digestion(TAD), or liquid composting, is a versatile new process for the treatment and stabilization of high strength wastes of liquid or, perhaps more importantly, slurry consistency. The pattern of inactivation of various pathogenic and indicator organisms was studied using batch digestions under conditions that may be expected to be found in full-scale TAD processes. Rapid inactivation of test populations occurred within the first 10 min from the start of digestion. The inactivation rate was slightly lower when digestions were conducted below 60 degrees C. In some instances, a 'tail' was apparent, possibly indicating the survival of relatively resistant sub-populations particularly in the case of Serratia marcescens and Enterococcus faecalis, or of clumping or attachment of cells to particulate materials. The effect of pH on the inactivation of the test populations depended on the temperature of digestion, but varied with the test population. At 55 degrees C Escherichia coli was more sensitive to temperature effects at pH 7 than at pH 8, but was more sensitive at pH 8, 60 degrees C. The reverse was the case at 60 degrees C for Ent. faecalis. An increase in the solid content of the digesting waste caused a progressive increase in the protection of test organisms from thermal inactivation. Challenging a TAD process with test strains allows (via estimation of D-values) a quantification of the cidal effects of such processes, with a view to manipulating process variables to enhance such effects.

Agriculture↗
PubMed1996

An improved method of partially digesting plant megabase DNA suitable for YAC cloning: application to the construction of a 5.5 genome equivalent YAC library of tomato.

An improved method for preparing partially digested tomato DNA has been developed, that is suitable for YAC cloning. It involves (i) isolation of high molecular-weight DNA from agarose-embedded leaf protoplasts, (ii) controlled partial digestion in situ using EcoRl endonuclease in the presence of EcoRI methylase (M. EcoRl), and (iii) fractionation of the partial digest on a Clamped Homogeneous Electric Fields (CHEF) gel. Unlike methods commonly used for generating partial digests, the present method allows one to produce digests in which the bulk of restriction fragments are of the desired size. Use of these partial digests in constructing YAC libraries of the tomato lines Moneymaker-Cf4 and VFNT Cherry resulted in libraries (total 21,060 clones, 5.5 genome equivalents) in which 80% of the YACs have inserts between 200 and 600 kb. Both libraries have been screened with selected RFLP markers linked to the Cladosporium fulvum Cf4 locus on chromosome 1, using a three-dimensional PCR-based screening technique. To this end, the RFLP markers have been sequenced to allow for the synthesis of specific primers. Thus, for each marker tested several YAC clones have been isolated, including a family of clones that carry leucine-rich repeat sequences located around the Cf4/Cf9 locus.

Base Sequence↗
PubMed1991

Digestibility of a nutritionally-balanced cassava (Manihot esculenta Crantz) diet and its effect on growth in young male dogs.

Experiments were carried out to study the digestibility of a cassava (gari) diet and its effect on growth in young male dogs. Three groups of dogs were fed on diets with rice (control), cassava (gari), and rice + cyanide respectively as the carbohydrate source. Each diet contained 130 g crude protein (nitrogen x 6.25)/kg, was supplemented with vitamins and minerals, and was fed for 14 weeks. Variables measured were body-weight gain, bone growth, plasma alkaline phosphatase (EC 3.1.3.1) activity, total serum 3,5,3'-triiodothyronine (T3) and some plasma free amino acids. The apparent digestibilities of dry matter, protein and fat were not significantly different in the three groups, but the digestibility of gari fibre was significantly lower than the digestibility of rice fibre when fed to dogs (P less than 0.05). Proximate analysis of the faeces showed that the group of dogs fed on the gari diet had faeces which had a significantly higher moisture content than the faeces of the other groups (P less than 0.05), and also a significantly higher fibre content (P less than 0.05). There was no significant difference in body-weight gain and bone growth between the control and gari-fed groups of dogs, but these variables were significantly lower in the dogs fed on the rice + cyanide diet (P less than 0.05). At the end of the 14-week experimental period total serum T3 and plasma alkaline phosphatase activity were not significantly different between the control group of dogs and the gari-fed group, but were significantly lower in the rice + cyanide group. Plasma free methionine, leucine, isoleucine and valine concentrations were higher in the rice + cyanide group of dogs than in the control group and the gari group, indicating that these amino acids were accumulating and not being utilized for protein synthesis and growth to the same extent in the rice + cyanide group of dogs as in the other groups. It was concluded that the digestibilities of cassava starch and rice starch were the same in the dog but that rice fibre was more digestible in the dog than cassava fibre. It was also concluded that growth proceeded normally when a balanced gari diet or a balanced rice diet containing 130 g crude protein/kg was fed to dogs, but growth was retarded when a balanced rice + cyanide diet containing 130 g crude protein/kg was fed to dogs because total serum T3 concentration became greatly depressed.

Alkaline Phosphatase↗