Amino acid analysis on polyvinylidene difluoride membranes

A procedure for the amino acid analysis of proteins electrotransferred to polyvinylidene difluoride (PVDF) membranes is described. The proteins are first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a PVDF membrane. After staining with Coomassie brilliant blue, the visualized protein bands are excised from the membrane. Each band is placed in a vial and subjected to gas-phase hydrolysis in 6 N HCl in a vacuum desiccator at 110 degrees C. The amino acids are extracted from the membrane into 0.1 N HCl/30% CH3OH and analyzed by reverse-phase HPLC using postcolumn o-phthalaldehyde-derivatizing reagent. The method was shown to give reproducible and reasonably accurate compositions for several proteins, as well as to provide an estimate of protein content. As little as 10 pmol of a 67-kDa protein can be determined.     

Direct carbohydrate analysis of glycoproteins electroblotted onto polyvinylidene difluoride membrane from sodium dodecyl sulfate-polyacrylamide gel.

A procedure for the carbohydrate analysis of glycoproteins electrotransferred to a polyvinylidene difluoride membrane is described. The glycoproteins (plant lectins, transferrin, and vitronectin) were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a membrane. Each of the glycoprotein bands visualized by staining with Coomassie brilliant blue R-250 was excised from the membrane and subjected to direct hydrolysis either in 2.5 M trifluoroacetic acid at 100 degrees C for 6 h for neutral sugars and hexosamines, or in 0.05 M H2SO4 at 80 degrees C for 1 h for sialic acids. The hydrolysate obtained was analyzed for neutral sugars, hexosamines, and sialic acids independently by three different systems of high-performance liquid chromatography. The analytical values were reproducible with reasonable accuracy and agreed with those expected with recoveries of 57-66%. The method was successfully applied to a mannose-specific lectin of Sophora japonica bark, which is composed of four different subunits that aggregate sugar specifically. Because the four subunits could be separated by SDS-PAGE alone, the method proved useful for determining their carbohydrate compositions. Three of them were shown to contain carbohydrates typical of N-linked oligosaccharides of plant origin, which agreed well with the results of the binding assay carried out on a membrane using various horseradish peroxidase-labeled lectins.     

Recovery of tryptophan in peptides and proteins by high-temperature and short-term acid hydrolysis in the presence of phenol

The addition of 3% (w/v) phenol to 6 M HCl largely prevented the destruction of tryptophan during rapid hydrolysis of peptides and proteins at 166 degrees C for 25 min or at 145 degrees C for 4 h. This hydrolysis procedure was advantageous for amino acid microanalysis using conventional high-performance liquid chromatography with a precolumn derivatization technique. The recovery of tryptophan from proteins was at least 80%. The addition of phenol also improved the recovery of methionine and carboxymethylcysteine. The amount of tryptophan in proteins electroblotted onto a polyvinylidene difluoride membrane was determined by this method.     

A rapid vapor-phase acid (hydrochloric acid and trifluoroacetic acid) hydrolysis of peptide and protein

A new method for the acid hydrolysis of protein is presented. Peptide bonds are cleaved by the action of an HCl/trifluoroacetic acid (TFA) vapor mixture. Contamination for the hydrolysis mixture is reduced to low levels (1-3 pmol). Recovery of hydrophobic amino acid is improved. Short reaction times are achieved and rapid removal of acids is facilitated. The reaction temperature is 158 degrees C for reaction times of 22.5 and 45 min with 7 M HCl and 10% TFA containing 0.1% phenol.     

Deblocking and subsequent microsequence analysis of N alpha-blocked proteins electroblotted onto PVDF membrane.

A method was developed for direct microsequencing of N alpha-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N alpha-Acetylated proteins (greater than 32 pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37 degrees C for 30 min. The protein was digested on the membrane with 5-10 micrograms of trypsin at 37 degrees C for 24 h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50 mU of acylamino acid-releasing enzyme at 37 degrees C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.     

Microsequence analysis of electroblotted proteins. II. Comparison of sequence performance on different types of PVDF membranes.

The influence of different types of polyvinylidene difluoride (PVDF) membranes on gas phase sequence performance has been evaluated. These PVDF membranes have been classified as either high retention (Trans-Blot and ProBlott) or low retention membranes (Immobilon-P) based on their ability to bind proteins during electroblotting from gels. Initial yields, repetitive yields, and extraction efficiency of the anilinothiazolinone amino acid derivatives have been compared for several standard proteins that have been either electroblotted or loaded onto PVDF membranes by direct adsorption. These results show that the major differences in initial sequence yields between membranes arise from differences in the amount of protein actually transferred to the membrane rather than sequencer-related factors. In contrast to several previous observations from other laboratories, more tightly bound proteins do not sequence with lower initial yields and initial yields are not affected by the ratio of surface area to protein. The stronger binding on high retention PVDF membranes does not adversely affect recoveries of difficult to extract, or very hydrophobic, amino acid derivatives. Several amino acids, especially tryptophan, are actually recovered in dramatically higher yield on high retention membranes compared with either Immobilon or glass filters. At the same time, the protein and peptide binding properties of high retention membranes will frequently improve the repetitive yield by minimizing sample extraction during the sequencer cycle. Stronger protein binding together with improved electroblotting yields offer substantially improved sequence performance when high retention PVDF membranes are used.     

Phosphoamino acid analysis of protein immobilized on polyvinylidene difluoride membrane

The direct analysis of phosphorylated proteins bound to polyvinylidene difluoride membrane (PVDFm) has been examined. Use of 14C-methylated marker proteins demonstrated that proteins electroblotted on PVDFm were quantitatively retained through a series of test conditions, which included 1 M hydroxylamine (25 degrees C, 30 min), 0.1 M NaOH (37 degrees C, 30 min), 0.1 M HCl (55 degrees C, 2 h), and 6 U/ml alkaline phosphatase (pH 9.5, 37 degrees C, 24 h). Approximately half the protein remained bound following 2-h treatment in 1 M KOH (55 degrees C). The same series of test conditions were employed to assess the stability of phosphorylated residues in 32P-labeled protein immobilized on PVDFm, in order to assign them as carboxyl-,N-, or O-linked groups. The properties of phosphorylated proteins as determined by this method were comparable to the properties that have been reported for soluble proteins. Use of the PVDFm immobilization step affords simplification of the experimental procedures and permits rapid, quantitative sample recovery using submicrogram quantities of protein. Further, the PVDFm-bound phosphoproteins could be subjected to partial acid hydrolysis directly on the membrane and required no further purification for subsequent identification of the labeled phosphohydroxyamino acids. Definitive identification of labeled phosphoserine residues in histone, phosphoserine and phosphothreonine residues in myelin basic protein and insulin receptor, and phosphotyrosine residues in autophosphorylated insulin receptor was accomplished with as little as 0.2 nCi in about 50 ng of phosphorylated protein.     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

Proteomic analysis of acrylamide gel separated proteins immobilized on polyvinylidene difluoride membranes following proteolytic digestion in the presence of 80% acetonitrile

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) combined with mass spectrometry (MS) is a highly accurate and sensitive means of identifying proteins. We have developed a novel method for digesting proteins on polyvinylidene difluoride (PVDF) membranes for subsequent matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis. After Tricine sodium dodecyl sulfate (SDS)-PAGE, separated proteins were electroblotted onto PVDF membranes in a semidry discontinuous buffer system, visualized by staining with Coomassie Blue, excised, digested with trypsin or lysC in 80% acetonitrile, and then analyzed by MALDI-TOF MS. This method has several advantages over in-gel digestion in terms of sample handling, sensitivity, and time. We identified 105 fmol of Bacillus subtilis SecA and 100 approximately 500 fmol of standard proteins. We also analyzed the submembrane protein fraction solubilized by 1% n-dodecyl-beta-D-maltoside from B. subtilis membranes after separation by 2-D PAGE, and identified 116 protein spots. This method can detect proteins at the 10 approximately 50 fmol level by pooling more than ten identical electroblotted protein spots.     

Quantitation of proteins bound to polyvinylidene difluoride membranes by elution of coomassie brilliant blue R-250

A rapid and simple method for the quantitation of stained proteins bound to polyvinylidene difluoride (PVDF) membranes via the elution of Coomassie brilliant blue R-250 is described. A mixture of standard proteins was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. Spectrophotometric analysis of dye eluted from protein bands in the range of 0.5-10 micrograms gave a linear change in the absorbance at 595 nm. Maximal absorbance readings were attained following 5 min of dye elution, and the readings remained unchanged for elution times up to 60 min. The method requires no unusual reagents or equipment, is suitable for the analysis of multiple samples, and does not consume the protein in the process of quantitation. This technique provides a useful means for the quantitation of proteins bound to PVDF membranes prior to amino acid sequence determination, immunological analysis, or other biochemical characterizations.     

Sequential GC/MS analysis of sialic acids,monosaccharides, and amino acids of glycoproteins on a single sample as heptafluorobutyrate derivatives

A GC/MS procedure was developed for the analysis of all major constituents of glycoproteins. The rationale for this approach is that by using GC/MS analysis of the constituents as heptafluorobutyrate derivatives, it was possible to quantitatively determine the sialic acid, monosaccharide, fatty acids (when present), and the amino acid composition with the sample remaining in the same reaction vessel during the entire procedure. A mild acid hydrolysis was used to liberate sialic acids and was followed by formation of methyl-esters of heptafluorobutyrate (HFB) derivatives. After GC/MS analysis of sialic acids, the remaining material was submitted to acid-catalyzed methanolysis followed by the formation of HFB derivatives. After GC/MS analysis of the monosaccharides, the sample was supplemented with norleucine (as internal standard) and hydrolyzed with 6 M HCl followed by the formation of isoamyl-esters of HFB derivatives and GC/MS analysis. His and Trp residues were modified during the step of acid-catalyzed methanolysis, but the resulting derivatives were stable during acid hydrolysis and quantitatively recovered by GC/MS analysis. As a result, all constituents of glycoproteins (sialic acids, monosaccharides (or di- and trisaccharides) and amino acids) are identified in the electron impact mode of ionization and quantified using three GC/MS analysis in the same chromatographic conditions and using a limited number of reagents, a considerable advantage over previous techniques. This method is very sensitive, all data (qualitative and quantitative) being obtained at the sub-nanomolar level of initial material.     

Post-electrophoretic identification of oxidized proteins

The oxidative modification of proteins has been shown to play a major role in a number of human diseases. However, the ability to identify specific proteins that are most susceptible to oxidative modifications is difficult. Separation of proteins using polyacrylamide gel electrophoresis (PAGE) offers the analytical potential for the recovery, amino acid sequencing, and identification of thousands of individual proteins from cells and tissues. We have developed a method to allow underivatized proteins to be electroblotted onto PVDF membranes before derivatization and staining. Since both the protein and oxidation proteins are quantifiable, the specific oxidation index of each protein can be determined. The optimal sequence and conditions for the staining process are (a) electrophoresis, (b) electroblotting onto PVDF membranes, (c) derivatization of carbonyls with 2,4-DNP, (d) immunostaining with anti DNP antibody, and (e) protein staining with colloidal gold.     

Do polyamines contribute to plant cell wall assembly by forming amide bonds with pectins?

Two new reducing glycoconjugates [N-D-galacturonoyl-putrescinamide (GalA-Put) and N,N'-di-D-galacturonoyl-putrescinamide (GalA-Put-GalA)] and homogalacturonan-putrescine (GalAn-Put) conjugates were synthesised as model compounds representing possible amide (isopeptide) linkage points between a polyamine and either one or two pectic galacturonate residues. The amide bond(s) were stable to cold acid and alkali (2M TFA and 0.1M NaOH at 25 degrees C) but rapidly hydrolysed by these agents at 100 degrees C. The amide bond(s) were resistant to Driselase and to all proteinases tested, although Driselase digested GalAn-Put, releasing fragments such as GalA3-Put-GalA3. To trace the possible formation of GalA-polyamine amide bonds in vivo, we fed Arabidopsis and rose cell-cultures and chickpea internodes with [14C]Put. About 20% of the 14C taken up was released as 14CO2, indicating some catabolism. An additional approximately 73% of the 14C taken up (in Arabidopsis), or approximately 21% (in rose), became ethanol-insoluble, superficially suggestive of polysaccharide-Put covalent bonding. However, much of the ethanol-inextractable 14C was subsequently extractable by acidified phenol or by cold 1M TFA. The small proportion of radioactive material that stayed insoluble in both phenol and TFA was hydrolysable by Driselase or hot 6M HCl, yielding 14C-oligopeptides and/or amino acids (including Asp, Glu, Gly, Ala and Val); no free 14C-polyamines were released by hot HCl. We conclude that if pectin-polyamine amide bonds are present, they are a very minor component of the cell walls of cultured rose and Arabidopsis cells and chickpea internodes.     

Optimum conditions of autoclaving for hydrolysis of proteins and urinary peptides of prolyl and hydroxyprolyl residues and HPLC analysis

A method for urinary peptide(s) and protein hydrolysis, involving autoclaving at 15psi (121 degrees C) for 60min, is described. Using three candidate proteins (bovine serum albumin, casein and gelatin) and urine specimens, the effect of autoclaving with respect to the optimum time required for hydrolysis under both acidic (6N HCl) and alkaline (6N KOH) conditions was studied. Recoveries of total amino acids from proteins and urine hydrolysate(s) suggest that complete hydrolysis of proteins and urinary peptides could be achieved by autoclaving for 30-60min instead of 16h of incubation at 110 degrees C. Further, stability of some of the individual amino acids was also studied. The observed differential stability of amino acids under acidic and alkaline conditions, as demonstrated in this study by HPLC analysis, makes it imperative to choose the appropriate hydrolytic condition while studying the composition of any given amino acids in urinary peptide(s)/protein hydrolysates. Further, the finding that both Pro and Hyp were stable under alkaline conditions of hydrolysis by autoclaving renders this method suitable for assaying these two amino acids from urine hydrolysates, hence its utility in the study of urinary peptide derived Hyp and Pro in bone/cartilage disorders.     

Hydrolysis of bacterial wall carbohydrates in the microwave using trifluoroacetic acid

By and large, monosaccharide composition and linkage analyses of bacterial cell-surface carbohydrates are achieved by hydrolysis into the corresponding monomeric constituents, and characterization of these, or their derivatives, by chromatographic and spectrometric methods. Normally, these hydrolyses are carried out conveniently with trifluoroacetic acid (TFA) at high temperatures for long periods of time, for example, in 4M TFA at 100 degrees C for 5h in a heating block. In this study, using a closed-vessel system, we investigated the effectiveness and reliability of microwave-assisted TFA hydrolysis of bacterial lipopolysaccharides, capsule, and teichoic-acid polysaccharides that were variably composed of several glycoses. In all cases, we were able to establish that 5min of hydrolysis in the microwave at 120 degrees C with 4M TFA (measured pressure of 90psi) was sufficient time to obtain comparable results to those afforded by conventional hydrolysis. The same observation was made when fully methylated carbohydrates were hydrolyzed. The data obtained with our microwave system (Aurora Instruments MW600) showed that microwave-induced hydrolysis can be used with a high degree of confidence to carry out sugar composition analysis of complex bacterial glycans in markedly shorter periods of time. The results also suggested that non-thermal mechanistic factors must also be involved, at least in part, in accelerating the reaction rate of glycosidic hydrolysis.     

Acid hydrolysis of protein in a microcapillary tube for the recovery of tryptophan

The acid hydrolysis of proteins was miniaturized and simplified by employing microcapillary tubes (100 microl in volume) with 6 M HCl containing 1% 2-mercaptoethanol and 3% phenol for an amino acid compositional analysis. The method not only eliminated the laborious evacuation step for the hydrolysis tube but also decreased the destruction of tryptophan during hydrolysis. The recovery of tryptophan was 79% by acid hydrolysis at 145 degrees C for 4 h. Since the acid mixture could be removed under vacuum, the hydrolysate was subjected to an amino acid analysis without neutralization or dilution.     

Purification of rabbit and human serum paraoxonase.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.     

Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes

Small amounts (7-250 pmol) of myoglobin, beta-lactoglobulin, and other proteins and peptides can be spotted or electroblotted onto polyvinylidene difluoride (PVDF) membranes, stained with Coomassie Blue, and sequenced directly. The membranes are not chemically activated or pretreated with Polybrene before usage. The average repetitive yields and initial coupling of proteins spotted or blotted into PVDF membranes ranged between 84-98% and 30-108% respectively, and were comparable with the yields measured for proteins spotted onto Polybrene-coated glass fiber discs. The results suggest that PVDF membranes are superior supports for sequence analysis of picomole quantities of proteins purified by gel electrophoresis.     

Extraction of protein and amino acids from deoiled rice bran by subcritical water hydrolysis

This study investigated the production of value-added protein and amino acids from deoiled rice bran by hydrolysis in subcritical water (SW) in the temperature range between 100 and 220 degrees C for 0-30 min. The results suggested that SW could effectively be used to hydrolyze deoiled rice bran to produce useful protein and amino acids. The amount of protein and amino acids produced are higher than those obtained by conventional alkali hydrolysis. The yields generally increased with increased temperature and hydrolysis time. However, thermal degradation of the product was observed when hydrolysis was carried out at higher temperature for extended period of time. The highest yield of protein and amino acids were 219 +/- 26 and 8.0 +/- 1.6 mg/g of dry bran, and were obtained at 200 degrees C at hydrolysis time of 30 min. Moreover, the product obtained at 200 degrees C after 30 min of hydrolysis exhibited high antioxidant activity and was shown to be suitable for use as culture medium for yeast growth.     

Diagnostic methods for the determination of iduronic acid in oligosaccharides

A high-performance liquid chromatography (HPLC) method with pulsed amperometric detection (PAD) was used for the determination of the acid hydrolysis products of L-iduronic acid containing oligosaccharides isolated from biological sources. This HPLC-PAD method was compared with gas chromatographic (GLC) methods. Since acid hydrolysis of oligosaccharides can produce a number of products, several uronic acid derivatives were prepared by chemical synthesis. These well characterized standards in conjunction with mass spectrometry allowed for the identification of most of the products of methanolysis or hydrolysis of glycosamino-glycans, which included chondroitin sulfates A and B (dermatan sulfate), heparin, and hyaluronic acid. (4 M) HCl in methanol 100 degrees C for 24 h was found to be optimum for GLC and 1 M aqueous HCl for 4 h at 100 degrees C for HPLC-PAD. All of the monosaccharides, hexosamines, and uronic acids could be separately identified in a single chromatographic step using either technique. Good resolution, high sensitivity (low microgram samples) and rapid analysis makes these methods particularly useful for the determination of small amounts of glycosaminoglycans and other glycoconjugates found in samples isolated from biological sources. These two techniques are specifically designed to allow the qualitative determination of the carbohydrate content and composition of samples whose carbohydrate composition and content is completely unknown.