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PubMed2003

Electrospray ionization from a droplet deposited on a surface-modified glass rod.

This paper reports development of a non-mechanical electrospray ionization (ESI) method to generate electrospray from a droplet deposited on an optical fiber coated with a thin gold or Nafion film. Modification of the surface of the optical fiber in this manner increases its wettability, such that a droplet of the aqueous sample solution can adhere sufficiently strongly to the tip of the fiber. The aqueous sample solution was deposited near the tip of the fiber with a micropipette. When a high voltage (2,000 V) was applied to the fiber by electrical connection through the gold film, the sample solution moved and hung at the tip of the fiber. Simultaneously, ESI was generated from the sample droplet. Multiply charged peptide and protein ions were detected by connecting the ESI source to a quadrupole mass analyzer.

Angiotensin I↗
PubMed1994

Matrix-assisted laser desorption/ionization mass spectrometry of restriction enzyme-digested plasmid DNA using an active Nafion substrate.

Matrix-assisted laser desorption/ionization using a 3-hydroxypicolinic acid matrix from an active Nafion substrate has been used for detection of restriction enzyme-digested double-stranded plasmid DNA using time-of-flight mass spectrometry. DNA strands of up to 267 base pairs were detected with minimal sample purification, although only as species corresponding to single-stranded DNA.

Base Sequence↗
PubMed2002

Microwave-assisted protein staining: mass spectrometry compatible methods for rapid protein visualisation.

The effects of microwave irradiation on the staining of electrophoresed and electroblotted proteins have been assessed using currently available detection methods. Although the absorption of microwave radiation was found to be uneven, band intensity following microwave-assisted protein staining (MAPS) was comparable and in some cases exceeded the intensity of the bands visualised by the original staining methods. It was found that microwave treatment drastically reduced the duration of the staining protocols for visualisation of the proteins separated by both one- and two-dimensional electrophoresis. Application of MAPS methods did not affect peptide mass fingerprinting analysis by mass spectrometry and subsequent identification of the protein by database searching. The peptide mass maps corresponding to the proteins visualised using both the conventional and MAPS methods did not show significant difference in signal/noise ratio. Moreover, it appeared that microwave treatment of the gels resulted in the increased recovery of the peptides following in-gel trypsin digestion. Briefly, microwave-assisted protein staining methods were rapid, compatible with mass spectrometry and were equally effective on thin (0.75-mm) and thick (1.5-mm) gels (such as those used in 2D electrophoresis).

DNA Fingerprinting↗
PubMed1993

An assay to detect glycoproteins that contain mannose 6-phosphate.

We have developed a broadly applicable solid phase assay for quantitative and qualitative analysis of phosphorylated lysosomal enzymes and other glycoproteins that contain mannose 6-phosphate (Man6-P) residues. Samples are immobilized on membranes and proteins that contain Man6-P are detected using an iodinated, soluble form of the cation-independent Man6-P receptor. The assay is highly specific, sensitive, and linear over 3 orders of magnitude. The technique has two general applications. First, it can be used in a dot blot format to analyze large numbers of samples for total levels of Man6-P glycoproteins. Second, it can be used in a western blot format to identify Man6-P glycoproteins that have been fractionated by polyacrylamide gel electrophoresis. Specific applications of this novel assay include structural characterization of glycoproteins, diagnosis of mucolipidosis II and III, and screening of pharmaceuticals for lysosomotropic activity. Other potential applications are discussed.

Animals↗
PubMed1993

Covalent attachment of peptides to membranes for dot-blot analysis of glycosylation sites and epitopes.

Noncovalent binding of proteins to membranes is often employed for dot-blot analysis with various visualization techniques. These techniques are usually not applicable to peptide dot-blot analysis due to peptide wash-off during the staining procedure. As exemplified with a synthetic peptide and peptides produced by proteolysis of a protein, it is possible to achieve efficient covalent attachment to Immobilon-AV membranes. The utility of this membrane has been demonstrated with immunostaining and carbohydrate staining procedures.

Amino Acid Sequence↗
PubMed1993

In vivo pulse labeling of proteins in attached leaves with radioactive amino acids.

The pulse labeling of proteins in attached leaves by the incorporation of radioactive amino acids can be a valuable approach to study a wide range of topics pertaining to plant gene expression. Transient changes in the rate of net protein synthesis can be measured since pulse labeling permits discrimination between proteins actively synthesized during the labeling interval from those accumulated at earlier times. Specific procedures for a rapid and convenient method of in vivo pulse labeling are outlined which we believe could be broadly useful in plant biological research. A particularly important technical feature of the protocol is electroblotting of radioactive proteins from the sodium dodecyl sulfate acrylamide resolving gel matrix to polyvinylidene difluoride transfer membrane. This step permits a single polyacrylamide slab gel to be used for autoradiography, immunoblot analysis, and protein staining, thereby greatly facilitating comparison among these complementary techniques and resulting in a significant savings in time and radioactive sample. Central considerations concerning the quantitation and interpretation of in vivo pulse labeling data are discussed.

Amino Acids↗
PubMed1993

A semidry electroblotting system efficiently transfers both high- and low-molecular-weight proteins separated by SDS-PAGE.

A general procedure is described for the simultaneous recovery by electroblotting on nitrocellulose or polyvinylidene difluoride (PVDF) membranes of both high- and low-molecular-weight proteins already separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is based on the use of an original set of buffers which creates a stable pH boundary between the two faces of the blotted membrane. The gel to be transferred is placed on the basic side (pH 8.4) of this boundary, which also contains sodium dodecyl sulfate. Under these conditions even the high-molecular-weight proteins are efficiently eluted out of the acrylamide. Their migration is then stopped on the membrane, due to the change of pH with the acid side (pH 3.8) of the boundary, and the presence of methanol in it. This increases the adsorption on the membrane of the low-molecular-weight polypeptides which have a low affinity for nitrocellulose or PVDF. The procedure permits a quantitative transfer of complex samples which contain poorly soluble proteins or with molecular masses outside the range of 20-70 kDa, like grain storage proteins of cereals.

Buffers↗
PubMed1993

High-sensitivity gas phase sequence analysis of proteins and peptides on PVDF membranes using short cycle times.

An optimized sequencer program with a cycle time of 38 min which is specifically tailored for analysis using polyvinylidene difluoride (PVDF) membranes has been developed. The program was developed using a pulsed liquid-phase instrument which was converted to gas-phase acid delivery. Gas-phase acid delivery minimized sample extraction from PVDF membranes and improved tryptophan yields in at least some cases. Other modifications which contributed to reliable high sensitivity sequencer performance included use of a Blott cartridge, substitution of ethyl acetate:heptane (1:1, v/v) instead of butyl chloride as the extraction solvent, use of a modified 100-microliters injection loop with an internal restrictor to reliably inject nearly 90% of the sample, and an HPLC gradient which resolved tryptophan from diphenylurea. These shortened cycle times were achieved at the conventional gas-phase reaction temperature. A slight increase in lag or carryover at prolines was compensated by reduced background from nonspecific acid cleavage which facilitated extended and/or high sensitivity sequencing of large proteins. Reproducible high initial and repetitive cycle yields were obtained with a wide range of experimental peptides which were electroblotted from either 1D or 2D polyacrylamide gels onto high retention PVDF membranes. Initial yields of the majority of the experimental samples analyzed with this program were less than 5 pmol. In addition, most samples with initial yields below 1-2 pmol yielded sufficient sequence information to identify the protein by comparison to protein sequence data-bases or to design oligonucleotide probes.

Amino Acid Sequence↗
PubMed1993

Pyridylethylation of cysteine residues in proteins.

A procedure is described for using an inert polyvinylidene difluoride type membrane (e.g., ProBlott) as a matrix for on-membrane gas-phase pyridylethylation and as a desalting (cleanup) support following the solution phase pyridylethylation of cysteine residues in proteins. As a desalting support for 200 pmol of cardiotoxin, the membrane-derived sample gave a better recovery of phenylthiohydantoin (PTH)-amino acids during N-terminal sequencing compared to the sample obtained by rpHPLC. Using on-membrane gas-phase pyridylethylation, followed by replacement of glass-fiber filter disc and membrane washing/drying in vacuo, it was possible to eliminate the broad background peak that coeluted with PTH-Ala in the HPLC chromatogram completely. Using this modified procedure, there was no evidence of N-terminal alkylation of the amino-termini of proteins.

Alkylation↗
PubMed1994

A method for high-performance sequence analysis using polyvinylidene difluoride membranes with a biphasic reaction column sequencer.

Methods have been developed for high-sensitivity sequence analysis of proteins electroblotted onto polyvinylidene difluoride (PVDF) membranes using a Hewlett-Packard G1005A protein sequencer. This sequencer normally uses a biphasic (hydrophobic/hydrophilic) reaction column which was designed to accommodate loading and cleanup of samples from diverse solutions. However, the standard column, programs, and chemistry were not designed to accommodate PVDF, which has become a common sequencing support. In this study, a systematic evaluation of the suitability of this sequencer for analysis using PVDF bound samples was performed and included evaluation of: different wash and extraction solvents, multiple programming changes, two alternative formulations of coupling reagents, and the effect of direction for solvent and reagent deliveries. High-performance analysis of PVDF bound samples was achieved by: using a modified reaction column with an empty hydrophobic (top) half of the column module, program modifications for the reaction column and converter, substitution of ethyl acetate for the standard S2/3 extraction solvent and using prototype Version 2.0 formulations of the coupling reagents, R1 and R2. High-performance sequence analyses of experimental samples electroblotted from either 1D or 2D gels onto high-retention PVDF membranes were obtained with a 41-min cycle time, including experimental samples with initial coupling yields < 2 pmol. Routine sequencer performance was comparable to, or slightly better than, a conventional gas-phase sequencer which had been previously optimized by us for high-performance sequence analysis of electroblotted samples in the low pmol range.

Amino Acid Sequence↗
PubMed1994

Derivatization of polyvinylidene difluoride membranes for the solid-phase sequence analysis of a phosphorylated sea urchin embryo histone H1 peptide.

We describe a simple and effective chemical protocol for attaching polyallylamine to polyvinylidene difluoride (PVDF) membranes. The underivatized PVDF discs were first etched in 0.5 M alcoholic KOH for 1.5 min and subsequently reacted with polyallylamine of high molecular weight under alkaline conditions. The covalently attached amino groups were reacted with 1,4-phenylene diisothiocyanate (DITC), thus converting the amino-modified PVDF discs to DITC-functionalized discs. The substitution level of the DITC-functionalized discs was found to be lower than that of the equivalent discs available commercially. Lysine-containing polypeptides were immobilized on these DITC-membrane discs and then subjected to either solid-phase manual Edman degradation or automated gas-phase sequencing. The method was found to be well suited for the sequencing of 32P-labeled phosphopeptides by employing the manual solid-phase technique.

Amino Acid Sequence↗
PubMed1994

An improved procedure for enzymatic digestion of polyvinylidene difluoride-bound proteins for internal sequence analysis.

An improved and simplified procedure for enzymatic digestion of proteins bound to polyvinylidene difluoride (PVDF) membranes for obtaining internal protein sequence data is presented. This improved procedure is compatible with various enzymes (trypsin, endoproteinase Lys-C, endoproteinase Glu-C, and clostripain) and is performed in the presence of 1% hydrogenated Triton X-100 (RTX-100)/10% acetonitrile/100 mM Tris, pH 8.0, followed by microbore HPLC purification of the recovered peptides. Previously published techniques required treatment of the PVDF-bound protein with polyvinylpyrrolidine M(r) 40,000 (PVP-40) prior to digestion, in order to prevent adsorption of the enzyme to the membrane. Unfortunately, contaminants produced from residual PVP-40 interfere with subsequent peptide mapping. We have found that when RTX-100 is used in the digestion buffer, no pretreatment of the PVDF-bound protein with PVP-40 is necessary. Advantages of this improved (one-step) procedure over the two-step PVP-40 procedure are (a) the elimination of undesirable contaminants associated with PVP-40, (b) a decrease in the time required for the technique, and (c) a reduction in sample manipulation. Peptide maps and recoveries from PVDF-bound standard proteins (4 micrograms each) enzymatically digested with this one-step method are compared with those obtained from the standard PVP-40 method. In addition, peptide maps and internal sequence data from low-level quantities of unknown proteins enzymatically digested with the improved procedure are presented. To date, this improved, one-step procedure has been successfully applied to 52 PVDF-bound unknown proteins (0.7-10 micrograms) of varying molecular weight (19-300 kDa) for which internal sequence data were obtained.

Amino Acid Sequence↗
PubMed1994

Determination of platelet-activating factor acetylhydrolase activity by blotting, beta-radioluminescence, and ultrahigh-sensitivity television camera detection.

A simple method for detecting the 1-alkyl-2-[3H]-acetyl-sn-glycero-3-phosphocholine ([3H]acetyl-PAF)-hydrolyzing activity of serum proteins is described. These were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The assay involves measurement of the beta-radioluminescence of [3H]acetyl-PAF on the PVDF membranes with an ultra-high-sensitivity TV camera system. Enzyme activity is detected as a decrease in beta-radioluminescence of [3H]-acetyl-PAF on the blotted membranes, since [3H]acetyl-PAF remains on the membrane but the [3H]acetate released by the enzyme reaction detaches from the membrane. This method has been used to identify PAF acetylhydrolase and phospholipase A2.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1994

Multiple use of blotted polyvinylidene difluoride membranes immunostained with nitro blue tetrazolium.

A simple procedure for reusing polyvinylidene difluoride membranes with bound proteins reacted with antibodies and developed by the nitro blue tetrazolium method (M. S. Blake, K. H. Johnston, G. J. Russell-Jones, and E. C. Gotschlich (1984) Anal. Biochem. 136, 175-179) is described. The blue diformazan precipitate produced by the reduction of the nitro blue tetrazolium salt was dissolved by soaking the membrane in dichloro- or trichloromethane for 10-60 min at room temperature. After equilibration in methanol and then in distilled water the first and second antibodies were removed by incubating the membrane in 0.2 M glycine-HCl buffer, pH 2.2, for 90 min at room temperature. These cleaned membranes could be successively reused with different monoclonal antibodies or preimmune serum up to 21 times. The intensity of the spots or electroblotted bands and their specific reaction pattern with different monoclonal antibodies remained unchanged, and no background was observed when the membranes were reacted with preimmune serum. This procedure makes it possible to reuse membranes freshly developed or stored for long periods without loss of resolution.

Membranes, Artificial↗
PubMed1994

Blotting of glycolipids and phospholipids from a high-performance thin-layer chromatogram to a polyvinylidene difluoride membrane.

A simple method of blotting glycosphingolipids from a high-performance thin-layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane is described. The developed HPTLC plate is dipped in a solvent mixture (isopropanol/0.2% CaCl2/methanol, 40/20/7 by volume) for blotting, after which first a PVDF membrane and then a glass microfiber filter is placed on the plate. The assemblage then is pressed for 30 s with heating. Most of the glycosphingolipids that are separated on the HPTLC plate can be blotted quantitatively and detected with the reagents used on the plate. Detection of the glycosphingolipids on the membrane was confirmed to be more sensitive than that on the HPTLC plate by both chemical visualization and immunological staining. The glycosphingolipids blotted on the membrane could be reextracted. Blotting of phospholipids can be done by the same method. The results suggest that this method can be used in the purification and characterization of glycosphingolipids and in the detection of proteins which recognize glycosphingolipids and phospholipids.

Animals↗
PubMed1994

Sequence analysis of lantibiotics: chemical derivatization procedures allow a fast access to complete Edman degradation.

Lantibiotics are antibiotic peptides produced via ribosomal synthesis of precursor proteins by gram-positive bacteria. They contain various unusual post-translational modifications, which include the formation of sulfide rings by lanthionine or beta-methyllanthionine, and 2,3-didehydroamino acids. The N-terminus may be blocked by a 2-oxobutyryl group and the C-terminus may be inaccessible in some of the lantibiotics. Due to these modifications the analysis of such peptides is very tedious. Chemical modifications using an ethanethiol-containing reaction mixture and/or trifluoroperacetic acid treatment were used to solve these analytical problems. Investigating the tetracyclic 22-peptide gallidermin and the N-terminally blocked tricyclic 34-peptide Pep5 as examples, a novel access to the primary structure of lantibiotics is demonstrated.

Amino Acid Sequence↗
PubMed1994

A simple and quantitative purification of glycosphingolipids and phospholipids by thin-layer chromatography blotting.

A new and simple method for purifying glycosphingolipids and phospholipids by using "TLC blotting" was established. Glycosphingolipids separated by two-dimensional thin-layer chromatography (TLC) were made visible with primuline reagent, and then bands were marked with a drawing colored pencil. The glycosphingolipids that separated on the HPTLC plate were transferred by TLC blotting to a polyvinylidene difluoride membrane together with the color marks. The marked areas were excised after which their glycosphingolipids were extracted and monitored by TLC. By this method, 20 glycosphingolipids showing homogeneous bands on a HPTLC plate were isolated from the neutral glycosphingolipid fraction of human meconium. Moreover, 10 kinds of acidic glycosphingolipids were purified as homogeneous bands from the bovine acidic glycosphingolipid fraction. The yields of glycosphingolipids (13 different ones) ranged from 68 to 92%, the mean value being 82.3%. The glycosphingolipids were confirmed to be purified as intact forms by mass spectrometric analysis and chromatographic mobilities on a HPTLC plate. The same procedure could also be used to purify phospholipids.

Animals↗