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.     

Microsequence analysis of peptides and proteins. VIII. Improved electroblotting of proteins onto membranes and derivatized glass-fiber sheets

We have quantitatively examined the various parameters affecting the electrotransfer and sequence analysis of proteins from sodium dodecyl sulfate (SDS) gels to derivatized glass fiber paper or to polyvinyldifluoride (PVDF) membranes. Transfer yields in the range of 90-95% can be obtained for proteins in the molecular weight range of 10-90 kDa for transfer from 12% SDS gels to glass fiber paper derivatized with either QAPS (N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride) or APS (aminopropyltriethoxysilane). In order to achieve these yields, it was necessary to modify the conditions described by R. Aebersold et al. (J. Biol. Chem. 261, 4229-4238, 1986). We activated the glass fiber paper with dilute ammonia water and derivatized the activated glass fiber paper with QAPS and APS in anhydrous solvents which were allowed to slowly absorb moisture during the derivatization process. The transfer yield varied with transfer time versus molecular weight of the protein for a given percentage gel. Shorter transfer times and higher yields were obtained for higher molecular weight proteins on 8% gels. Lower molecular weight protein gave higher yields from 12% gels under similar transfer conditions. Sequencing yields of the transferred proteins were in the range of 40-80%, but a number of background peaks were observed on HPLC analysis of the phenylthiohydantoin amino acid derivatives. Transfer yields in the range of 85-95% were observed for similar experiments with PVDF membranes. In order to achieve these yields, it was necessary to modify the conditions described by P. Matsudaira (J. Biol. Chem. 262, 10035-10038, 1987). A lower voltage and longer transfer times gave higher transfer yields. In order to achieve consistently high transfer yields, it was also necessary to precoat the PVDF membranes with Polybrene. The PVDF membranes were cut into approximately 1-mm-wide strips and inserted into a continuous flow reactor (J. E. Shively, P. Miller, and M. Ronk, Anal. Biochem. 163, 517-525, 1987) for sequence analysis. Overall yields of samples loaded onto gels, electrotransferred to Polybrene-coated PVDF membranes, and sequenced ranged from 50-60% for beta-lactoglobin (10-50 pmol loaded onto SDS gels) to 20-30% for bovine serum albumin and soybean trypsin inhibitor (50 pmol loaded onto SDS gels). A comparison of the two methods shows clear advantages for the PVDF membranes over the derivatized glass fiber paper, including the ability to directly sequence the Coomassie blue-stained PVDF membranes, and the lower backgrounds observed on subsequent sequence analysis.     

Microsequence analysis of winged bean seed proteins electroblotted from two-dimensional gel

Electroblotting method employing a semidry blotting apparatus for the subsequent protein microsequence analysis (Hirano, 1987) was improved. This method is convenient and allows rapid and efficient transfer of the proteins from a polyacrylamide gel (1 mm thick) onto the Polybrene-coated glass-fiber sheet or polyvinylidene difluoride membrane filter in only 20 min. The electroblotted proteins could be sequenced directly with the gas-phase protein sequencer at a 20-pmole level. This method was applied to the sequence analysis of winged bean seed proteins. A portion of the crude extracts from only one-twentieth of a seed of the winged bean was separated by two-dimensional polyacrylamide gel electrophoresis and electroblotted, and the N-terminal amino acid sequences of the blotted proteins were analyzed. The sequences of about 60% of the blotted major proteins, including nine Kunitz trypsin inhibitor-like proteins with heterogeneity in the N-terminal sequences, a protein that has a homologous sequence to the leghaemoglobin, nitrogen-fixing root nodule-specific protein, and a soybean basic 7S globulin-like protein could be easily identified.     

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.     

Solid-phase sequence analysis of proteins electroblotted or spotted onto polyvinylidene difluoride membranes

Electroblotted proteins noncovalently bound to polyvinylidene difluoride (PVDF) membranes are typically sequenced using adsorptive sequencer protocols (gas-phase or pulsed-liquid) that do not require a covalent linkage between protein and surface. We have developed simple chemical protocols where proteins are first electroblotted onto unmodified PVDF membranes, visualized with common protein stains, and then immobilized for solid-phase sequence analysis. Adsorbed, stained proteins are first treated with phenylisothiocyanate (PITC) to modify alpha and epsilon amines. The protein is then overlayed with a solution of 1,4-phenylene di-isothiocyanate (DITC), followed by a few microliters of a basic solution containing a poly(alkylamine). As the polymer dries onto the surface both polymer and remaining protein amino groups are crosslinked by DITC. The protein is thus immobilized to the membrane surface by entrapment in a thin polymer coating. The coating is transparent to the degradation chemistry, and extensive enough to remain immobilized even in the absence of any covalent link between polymer and surface. Partial modification with PITC allows for identification of N-terminal and internal lysine residues during sequencing. The process was tested with a variety of poly(alkylamines), linear and branched, with molecular weights ranging from 600 to over 100,000. Proteins bound in this manner were successfully sequenced using covalent (solid-phase) sequencer protocols with cycle times as short as 26 min.     

Microsequence analysis of peptides and proteins : I. Preparation of samples by reverse-phase liquid chromatography

Reverse-phase supports for the separation of peptides and proteins are compared in two high-performance liquid chromatographic systems. One uses a trifluoroacetic acid-acetonitrile solvent system with a 206-nm detector, and the other uses pyridine-formate or pyridine-acetate and 1-propanol with a postcolumn fluorescence detector. Each system was examined with RP8, RP18, and alkylphenyl supports. In most applications, the trifluoroacetic acid-acetonitrile solvent system used in conjunction with an alkylphenyl column performed best. The use of this system for the preparation of low-microgram amounts of samples for microsequence analysis is illustrated.     

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.     

Micro-determination of amino acid composition of proteins electroblotted onto polyvinylidene difluoride membranes

A method for determination of amino acid composition of proteins separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes is described. A single blotted band containing 50 to 200 pmoles of protein was cut out and submitted to acid hydrolysis with HCl followed by derivatization with phenylisothiocyanate. The amino acid derivatives were separated by reverse phase high-performance liquid chromatography. Bovine serum albumin, lysozyme, myoglobin, ovalbumin, soybean trypsin inhibitor and carbonic anhydrase were analyzed; the results revealed a good correspondence with reported values. This can be considered an analytical method to determine the amino acid composition of samples from microquantities of protein mixtures, particularly in those cases in which SDS-polyacrylamide gel electrophoresis is the most suitable separation system.     

Microsequence analysis of peptides and proteins. IX. Manual gas-phase microsequencing of multiple samples

A novel apparatus for performing manual gas-phase Edman chemistry on protein and peptide samples is described. Edman chemistry is performed in 6 to 10 Teflon continuous flow reactors (CFR), previously described by J.E. Shively et al. (1987) Anal. Biochem. 163, 517-529). The CFRs are packed with 10-15 mg of Polybrene-coated spherical silica (Porasil B, Waters Associates). The gas-phase coupling reagent and cleavage reagent are 5% aqueous triethylamine and anhydrous trifluoroacetic acid, respectively, delivered by a stream of argon gas. The delivery of the gas-phase reagents is manually controlled with Hamilton 3-way valves and 2-way valves, and that of the solvents, ethyl acetate and butyl chloride, by syringe pipetting. The average cycle time is 15-20 min for 6 to 10 samples run simultaneously. Conversion of the anilinothiazolinone to phenylthiohydantoin (PTH) amino acid derivatives is accomplished manually with 25% aqueous trifluoroacetic acid. The PTH amino acids are analyzed by reversed-phase HPLC using an autosampler for handling multiple samples. Excellent results were obtained in the 100-200 pmol range. Protein samples can be sequenced from 15-20 cycles, and peptide samples usually to the COOH terminus. Initial yields ranged from 30 to 60% and repetitive yields ranged from 90 to 96%. The sample washout and size of background peaks are significantly reduced, compared to older methods of manual sequence analysis. The yields and background signal to noise are comparable to automated gas-phase Edman chemistry. The improved manual Edman described represents a low cost alternative to automated sequence analysis, and has the advantage being able to process multiple samples simultaneously.     

Microsequence analysis of peptides and proteins. IX. An improved, compact, automated instrument

We describe the construction of an improved, compact protein sequencer with a vertical flow path and continuous flow reactor (CFR). Unique features include a hexagonal valve for six fluid inputs to the CFR, which connects vertically to a transfer valve that allows sample, reagent, and solvent input to a conversion flask (CF). The simplified CF contains only two inputs at the top, one for sample, reagent, and solvent input, and the other a vent. The CF drains from the bottom, connecting to a switching valve which allows either delivery to waste or to an on-line HPLC for the analysis of phenylthiohydantoin amino acid derivatives. Approximately 90% of the sample is analyzed by use of a sonic flow detector. The overall vertical flow path of the sequencer is about 16 cm. The size of the instrument (25 w x 38 x 44 d cm) is smaller than that of commercially available sequencers or HPLC systems. The performance of the instrument includes reduced background peaks and high-sensitivity sequence analysis at the 5-10 pmol level. The simplified sequencer is more economical and portable than conventional sequencers.     

Integrated stereological and biochemical studies of hepatocytic membranes. I. Membrane recoveries in subcellular fractions

Previous attempts to relate the structure and function of hepatocytic membranes have compared biochemical data of fractions to morphological data derived from either intact tissue or fractions. The effects of the original homogenization aside, biochemical recoveries comparing membrane marker enzymes of the homogenate to subsequent fractions suggest a general conservation of activity. A sterological study was undertaken to estimate membrane surface areas in the intact tissue, homogenate, and fractions of the same livers and then to test the comparability of these data with membrane marker enzymes by calculating both morphological and biochemical recoveries. The sterological data were corrected for errors due to section thickness and compression. The average total membrane sufrace area per 1 g of liver was 9.3 m2 in the intact tissue (T), 7.8 m2 in the homogenate (H), and 7.4 m2 in the fractions (F); recoveries for the membrane surface areas thus averaged 96% for the (F/H) and 81% for the (F/T) comparisons. In homogenate and fractions, the differentiability of membranes by morphological criteria was limited to rough- and smooth- surfaced membranes, as well as outer and inner mitochondrial membranes. The recoveries of rough-surfaced membranes were 101% for F/H and 92% for F/T; those of smooth-surface membranes were 89% for F/H and 107% for F/T. For mitochondrial membranes, a recovery of 100% for F/H was obtained, whereas it amounted to only 54% for F/T. With respect to F/H, the membrane recoveries compare well with the marker enzyme recoveries obtained biochemically. The extension of recovery calculations to the intact tissue (F/T) revealed satisfactory conservation of the procedures of homogenization and fractionation; it indicates, however, that a shift of a substantial part of mitochondrial membranes to the pool of unidentifiable smooth membranes may occur on homogenization.     

Microsequence analysis of peptides and proteins: IV. Structural studies on human leukocyte interferons

The sequence of the tryptic peptides of three major species of human leukocyte interferon was determined by microsequencing procedures. The peptides were aligned by comparison with the amino acid sequences predicted by the DNA sequences of recombinants containing leukocyte interferon-coding inserts. In addition, extended NH₂-terminal amino acid sequences of two human leukocyte interferons produced in Escherichia coli by recombinant DNA methodology are also reported. This report demonstrates application of microsequencing methodology to low nanomole and subnanomole amounts of proteins and peptides of biological interest.     

Microsequence analysis of peptides and proteins : III. Artifacts and the effects of impurities on analysis

Levels of contaminants in the parts-per-billion range can adversely affect amino acid microsequence analysis (low-nanomole to subnanomole range) in two ways; (a) contaminants in solvents used in the purification of proteins and peptides can derivatize reactive amino acids to form unusual products or react with free α-NH₂ groups to effectively prevent sequence analysis, and (b) contaminants in the reagents and solvents used in Edman chemistry can give spurious peaks on HPLC analysis of amino acid phenythiohydantoin derivatives or react with the phenylthiocarbamylpeptidyl derivatives to give lower initial and repetitive yields of the subsequent phenylthiohydantoin derivatives. Practical examples of these problems and their solutions are described. With proper care in the preparation of solvents and reagents for sample purification and Edman chemistry, microsequence analysis in the low-nanomole to subnanomole range can be made routine.     

A new siliconized-glass fiber as support for protein-chemical analysis of electroblotted proteins

A new hydrophobic glass-fiber support is presented, which is well suited to the electrophoretic transfer of proteins from polyacrylamide gels and subsequent protein-chemical analysis. Modified glass-fiber sheets are easily prepared by chemical reaction of the surface with poly(methyl-3,3,3-trifluoropropylsiloxane) in trifluoroacetic acid. The modification is stable during electroblotting, amino acid sequence analysis and hydrolysis. The siliconized glass fiber exhibits a high protein-binding capacity, allows the application of well-established staining procedures, and does not interfere with the analytical methods of modern protein chemistry at the low picomole level. Samples separated by electrophoresis and immobilized on hydrophobic supports fail to exhibit any detectable contamination in amino acid sequence analysis hence allowing the high performance of the available protein-chemical methods to be exploited.     

Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis

A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry.     

Microsequence analysis of peptides and proteins. VI. A continuous flow reactor for sample concentration and sequence analysis

We have designed and tested a continuous flow reactor (CFR) for microsequence analysis of peptides and proteins. The CFR forms the site for immobilization of the peptide or protein substrate and automated Edman chemistry. The CFR was constructed from 0.125-in.-o.d., 0.0625-in.-i.d. Teflon tubing (length 2-3 cm) containing 5-10 mg of Polybrene-coated, spherical, porous silica (100-200-micron particle size). The silica is retained in the CFR with porous Teflon filters (Zitex) at the bed bottom and optionally at the bed top. The i.d. of the CFR was selected for a tight press fit when 0.0625-in.-o.d. Teflon lines are inserted at the top and bottom of the CFR. This design allows the replacement of the existing cartridge/glass fiber disk found in conventional microsequencers with a CFR with a minimal amount of changes. The advantages of the CFR over the previous design include a lower background or noise level and no need to precycle Polybrene before sample application, and the entire unit is inexpensive and therefore disposable. We believe that the decrease in noise, especially the decrease in the commonly observed diphenylthiourea peak, is due to the more direct flow path and relative absence of unswept area in the CFR. Several standard peptides and proteins were sequenced in the CFR to demonstrate the improved results. A direct comparison to the cartridge/glass fiber disk design demonstrated less background and higher initial and repetitive yields for the CFR. An additional advantage is the ability to directly concentrate samples on CFRs containing reverse-phase packing. We have successfully concentrated 1.0-ml samples (200 pmol) onto 5 mg of octyldecylsilyl-derivatized silica in yields of 95-100%. The resulting samples were microsequenced after addition of Polybrene-coated silica to the CFR with high initial and repetitive yields. This methodology promises to improve sample handling and microsequence analysis of low picomole amounts of peptides and proteins.