Acetyl substitution patterns of amylose and amylopectin populations in cowpea starch modified with acetic anhydride and vinyl acetate

To study the effect of reagent type on the distribution pattern of acetyl groups in acetylated cowpea starch, amylose and amylopectin populations were isolated from the starch granules after modification to a low degree of substitution (DS < 0.1) with acetic anhydride and vinyl acetate, respectively. Slowly reacting reagent vinyl acetate resulted in higher DS values for the amylopectin populations when compared to the rapidly reacting reagent acetic anhydride. The two reagents had similar effects on the acetylation level of amylose, suggesting that the amorphous regions of granules were easily accessible for both reagents. The acetyl substitution patterns were analyzed by enzymatic degradation followed by characterization of the obtained fragments using chromatographic and mass spectrometric techniques. The distributions of acetyl groups along the amylose and amylopectin chains were more clustered for modification with vinyl acetate as compared with modification with acetic anhydride. Between the two acetylation types, pronounced differences in the acetyl substitution patterns were observed for the large fragments obtained after -amylase digestion; only slight differences were exhibited for the small fragments obtained by exhaustive enzymatic digestion of amylose and amylopectin populations.     

C-terminal sequencing by mass spectrometry: application to gelatine-derived proline-rich peptides

Protonated peptides derived from proline‐rich proteins (PRP) are often difficult to sequence by standard collision‐induced dissociation (CID) mass spectrometry (MS) due to preferential amide bond cleavage N‐terminal to proline. In connection with bovine spongiform encephalopathy regulations, proteolytic products derived from the PRP collagen have been suggested as markers for contamination of animal feedstuffs with processed animal protein (Fernandez Ocaña, M. et al., Analyst 2004, 129, 111–115). Herein, we report the identification of these marker peptides using the strategy of C‐terminal sequencing by CID MS from their sodium and lithium adducts. Upon fragmentation a new cationized peptide was produced that is one C‐terminal amino acid shorter in length. This dissociation pathway allowed for the facile identification of the C‐terminal residue by matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry. Each newly formed cationized peptide was further fragmented by up to seven stages of electrospray ionization ion trap MS. Proline‐rich C‐terminal sequence tags were established which permitted successful database identification of collagen alpha type I proteins.     

A modification of the acetic acid-urea system for use in microslab polyacrylamide gel electrophoresis

A modification of the S. Panyim and R. Chalkley (1969, Arch. Biochem. Biophys.130, 337) acetic acid-urea polyacrylamide gel system to include a stacking gel has been devised. The modification makes possible the use of acetic acid-urea gels in the microslab system of P. T. Matsudaira and D. R. Burgess (1978, Anal. Biochem.87, 386).     

Sequence similarity-driven proteomics in organisms with unknown genomes by LC-MS/MS and automated de novo sequencing

LC-MS/MS analysis on a linear ion trap LTQ mass spectrometer, combined with data processing, stringent, and sequence-similarity database searching tools, was employed in a layered manner to identify proteins in organisms with unsequenced genomes. Highly specific stringent searches (MASCOT) were applied as a first layer screen to identify either known (i.e. present in a database) proteins, or unknown proteins sharing identical peptides with related database sequences. Once the confidently matched spectra were removed, the remainder was filtered against a nonannotated library of background spectra that cleaned up the dataset from spectra of common protein and chemical contaminants. The rectified spectral dataset was further subjected to rapid batch de novo interpretation by PepNovo software, followed by the MS BLAST sequence-similarity search that used multiple redundant and partially accurate candidate peptide sequences. Importantly, a single dataset was acquired at the uncompromised sensitivity with no need of manual selection of MS/MS spectra for subsequent de novo interpretation. This approach enabled a completely automated identification of novel proteins that were, otherwise, missed by conventional database searches.     

A high-yield method for the isolation of hydrophobic proteins and peptides from polyacrylamide gels for protein sequencing

A methodological approach is described which allows the isolation of hydrophobic and hydrophilic proteins and peptides in high yield. The technique consists of (1) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) protein elution from polyacrylamide gels with an organic solvent mixture composed of formic acid/acetonitrile/isopropanol/H2O (50/25/15/10, v/v/v/v), and (3) purification of eluted proteins by size exclusion chromatography on a Superose 12 column using this organic solvent mixture as eluant. The efficiency of this technique was tested with radioactively labeled polypeptides. These proteins were reaction center from Chloroflexus aurantiacus, bacteriorhodopsin, halorhodopsin from Halobacterium halobium, bovine serum albumin, ovalbumin, alpha-chymotrypsinogen A, and cytochrome c. The elution recoveries from polyacrylamide gels were 77-95%; the final yield after chromatographic purification was still 67-76% (with one exception). Subsequent amino acid sequencing was possible without further sample treatment. The sensitivity of the method described was found to be at least 20-30 micrograms protein.     

Use of disc electrophoresis in polyacrylamide gel method for the separation of soluble proteins of vibrios]

The authors present the results of a comparative study of the protein spectra of 80 strains of various vibrios. Proteinograms were elaborated by the method of disc-electrophoresis in polyacrylamide gel for each strain and species. There were revealed 25 protein peaks with definite mobility coefficients. The number of peaks and their areas varied in different species. The qualitative and the quantitative differences between the vibrio species were established. Results of investigations demonstrated a possibliity of using the protein spectra for the differentiation of microorganisms.     

A method for the long-term preservation of polyacrylamide gel electrophoresis

A new, reversible method for drying polyacrylamide gel electrophoresis is reported. It was studied using proteins from the B17, B20, B21 and ATCC 8014 strains of Lactobacillus plantarum isolated from the brine of table olives. After electrophoretic analysis, the gels were dehydrated in a 95% ethyl alcohol solution and stored either long-term or for a few days, renatured and then subjected to analyses that included combination staining with Coomassie brilliant blue and silver, and western blotting. The immunological tests and electrophoresis performed with the enzymes β-glucosidase, alkaline phosphatase and peroxidase demonstrated that repeated dehydration and renaturation of the polyacrylamide gels does not denature the proteins. The method is simple to perform, inexpensive and does not require special equipment.     

Application of both high-performance liquid chromatography combined with tandem mass spectrometry shotgun and 2-D polyacrylamide gel electrophoresis for streptococcal exoproteins gave reliable proteomic data

Streptococci secrete a large number of exoproteins including virulence-associated toxins and enzymes. To construct a reliable database of streptococcal exoproteins, we integrated the results that were derived from two approaches: LC-based shotgun proteomic analysis and 2-D PAGE-based proteomic analysis. We identified 74 and 82 proteins by LC-based and gel-based analysis, respectively. Forty-five proteins were identified by both methods. In addition, two proteins, one identified by both methods and the other only by LC-based shotgun analysis, were newly annotated. We therefore found the importance of combinational analysis by the two methods for the construction of a more reliable database.     

Radiolabeling of proteins and viruses in vitro by acetylation with radioactive acetic anhydride.

We describe a convenient, rapid, and reproducible method for labeling proteins in vitro by acetylation with [3H] or [14-C]acetic anhydride dissolved in small amounts of anhydrous dioxane. The reaction is carried out at neutral pH and does not require the use of detergents, water-immiscible organic solvents, oxidizing, or reducing agents. Thus undesirable solvent-induced alterations in protein structure and biological activity are minimized. A method for calculating the specific activity of the protein and the efficiency of acetylation at known concentrations of protein and acetic anhydride is presented. Radioacetylated proteins were shown to be suitable for use as molecular weight calibration standards and as protein markers in polyacrylamide gel electrophoresis, gel filtration, and enzyme studies. Acetic anhydride was used to label intact oncornaviruses, which consist of a complex ribonucleo-protein core within a lipid envelope. Some of the viral lipid and all of the viral proteins, including the internal ones, were labeled without detectable alterations in viral morphology or buoyant density. This result suggests that acetic anhydride, evidently by virtue of its small size and neutral charge, penetrates freely throughout the viral membrane and core structures. The reactivity of RNA with acetic anhydride was less than 1% that of protein under similar reaction conditions.     

Sequential elution of proteins in small volumes by preparative polyacrylamide gel electrophoresis

A simple flexible method for separation of proteins by polyacrylamide gel electrophoresis and sequential elution into dialysis bags has been devised. The system was applied to isolation of three glycoproteins from the peritoneal fluid of mice bearing Ehrlich ascites tumor.     

A specific stain for sulfhydryl groups in proteins after separation by polyacrylamide gel electrophoresis

A new method is described for specifically staining protein sulfhydryl groups after the proteins have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in slab gels. The stain will detect as little as 0.25 microgram of lysozyme and 1 microgram of most other proteins; the range of sensitivity for a specific protein depending on its sulfhydryl content. Proteins with no cysteine residues (type I collagen) and glycoproteins do not cause spurious staining.     

Mass spectrometric sequencing of endotoxin proteins of Bacillus thuringiensis ssp. konkukian extracted from polyacrylamide gels

The amino acid sequences of the crystal proteins of Bacillus thuringiensis ssp. konkukian strain HL-47 are unknown. We used 1-D denaturing polyacrylamide electrophoresis, nano-ESI-Q-TOF-MS, and protein database searching to analyze these proteins. On SDS-PAGE gels, a preparation of purified crystal proteins exhibited 110, 102, 76, 55, 37, and 30 kDa protein bands. Immunoblotting of the gel with antiserum raised to this preparation revealed that four crystal proteins, of 110, 102, 55, and 37 kDa, reacted with the specific antiserum. The 102-kDa major protein reacted strongly. The other crystal proteins showed weak immunoreactivity. The 102 and 55 kDa proteins were analyzed by ESI-MS. The internal amino acid sequence of the 102-kDa major protein has similarity to the sequences of the surface layer protein of B. thuringiensis ssp. finitimus and B. anthracis. However, the internal amino acid sequences of the 55 kDa protein did not show any homology to proteins in the databases. Proteomic analysis of these proteins leads to the conclusion that the sequence data provided the protein databases of the crystal proteins of the konkukian ssp.     

Oven-drying method for polyacrylamide gel slab packed in cellophane sandwich

Polyacrylamide gel slabs can be dried quickly without elaborate tools and the results are similar or even better than those obtained with a commercial drying apparatus. The discontinuous, sodium dodecyl sulfate, and gradient polyacrylamide gel slabs yielded similar results regardless of the staining methods, e.g., Coomassie blue, periodate-Schiff's reagent, or ammoniacal silver.     

Database of two-dimensional polyacrylamide gel electrophoresis of proteins labeled with CyDye DIGE Fluor saturation dye

CyDye DIGE Fluor saturation dye (saturation dye, GE Healthcare Amersham Biosciences) enables highly sensitive 2-D PAGE. As the dye reacts with all reduced cysteine thiols, 2-D PAGE can be performed with a lower amount of protein, compared with CyDye DIGE Fluor minimal dye (GE Healthcare Amersham Biosciences), the sensitivity of which is equivalent to that of silver staining. We constructed a 2-D map of the saturation dye-labeled proteins of a liver cancer cell line (HepG2) and identified by MS 92 proteins corresponding to 123 protein spots. Functional classification revealed that the identified proteins had chaperone, protein binding, nucleotide binding, metal ion binding, isomerase activity, and motor activity. The functional distribution and the cysteine contents of the proteins were similar to those in the most comprehensive 2-D database of hepatoma cells (Seow et al.., Electrophoresis 2000, 21, 1787-1813), where silver staining was used for protein visualization. Hierarchical clustering on the basis of the quantitative expression profiles of the 123 characterized spots labeled with two charge- and mass-matched saturation dyes (Cy3 and Cy5) discriminated between nine hepatocellular carcinoma cell lines and primary cultured hepatocytes from five individuals, suggesting the utility of saturation dye and our database for proteomic studies of liver cancer.     

Visible labeling of proteins for polyacrylamide gel electrophoresis with dabsyl chloride

Several proteins, which are used as molecular weight markers in polyacrylamide gel electrophoresis, were reacted with dabsyl chloride. This labeled them deep orange and the chromophore attachment was stable throughout the electrophoretic procedure and fixation. Small amounts (10-50 micrograms) of the labeled proteins could be loaded onto gels and seen with the unaided eye so that the separation during electrophoresis could be followed. Dabsylation did not affect the mobility of the proteins. The location of the orange band gave a good indication of the position of the protein in the gel so that molecular weight estimations could be made during and immediately following electrophoresis.