This column highlights several recently published articles that are likely to be of interest to the readership of this newsletter. Articles are selected for listing and summarized by some members of the Editorial Board. Article summaries reflect their opinions and not necessarily those of the Association. We encourage ABRF Associates to forward information on articles they feel are important and useful to any member of the Editorial Board.
Amino Acid and Sequence Analysis
Wilkins, M.R., Ou, K., Appel, R.D., Sanchez, J.-C., Yan, J.X., Golaz, O., Farnsworth, V., Cartier, P., Hochstrasser, D.F., Williams, K.L. and Gooley, A.A. (1996) Biochemical and Biophysical Research Communications 221, 609-613. Rapid Protein Identification Using N-Terminal "Sequence Tag" and Amino Acid Analysis.
Describes the utility of combining amino acid composition analysis with other physical parameters to identify proteins from two-dimensional gels. Proteins bound to PVDF and stained with amido black are first subjected to 3 or 4 rapid cycles (23 minutes) of Edman degradation to provide amino-terminal "sequence tags". The same samples are then further characterized by automated Fmoc amino acid analysis. Software then uses the sequence tag, composition data, molecular weight, and isoelectric point to identify the protein by searching sequence databases. When using protein sequencers equipped with multiple sample cartridges, 5 to 10 samples can be analyzed each day.
Golaz, O., Wilkins, M.R., Sanchez, J.-C., Hochstrasser, D.F. and Williams, K.L. (1996) Electrophoresis 17, 573-579. Identification of proteins by their amino acid composition: An evaluation of the method.
Companion article from the same group emphasizing critical parameters involved in amino acid analysis, such as accuracy, reproducibility, and sources of contamination. Human keratin is singled-out as a common contaminant. Because of contamination from the membrane, and based on recoveries after hydrolysis and extraction from the membrane, amino acids are categorized as "unreliable" (H, G, Y, M, K, F) or "reliable" for weighting purposes in the searching software.
Yan, J.X., Wilkins, M.R., Ou, K., Gooley, A.A., Williams, K.L., Sanchez, J.-C., Golaz, O., Pasquali, C. and Hochstrasser, D.F. (1996) Journal of Chromatography A 736, 291-302. Large-scale amino-acid analysis for proteome studies.
More from the same group, this one emphasizing the critical first steps sampling handling and acid hydrolysisin the method. These steps can be streamlined to minimize sample manipulation by using a single vial for hydrolysis, extraction, and Fmoc-derivatization. The authors claim "their approach is the most cost-effective and time-effective first step in protein screening for a large-scale proteome project."
Werner, W.E., Grimely, C. and Yuan, P.-M. (1996) Analytical Biochemistry 237, 146-149. Effect of Polybrene on the N-Terminal Sequencing of Peptides Bound to Polyvinylidene Difluoride Membranes.
Addresses the commonly held belief that polybrene is needed to successfully sequence PVDF-bound peptides. ABI ProSorb cartridges were used to wick 10-pmol amounts of peptide samples to PVDF membranes. For 4 moderately hydrophilic peptides 8-12 residues long, all 36 residues were assigned when polybrene was used but only 29 when polybrene was omitted. For 3 longer peptides, the success rate was 90 of 92 residues with polybrene and 57 of 92 without.
Burkhart, W., Moyer, M.B., Bailey, J.M. and Miller, C.G. (1996) Analytical Biochemistry 236, 364-367. Electroblotting of Proteins to Teflon Tape and Membranes for N- and C-Terminal Sequence Analysis.
The same sample, blotted to Teflon and stained with sulforhodamine B, can be first amino-terminally sequenced (on an HP1005A instrument) and then subjected to carboxyl-terminal sequencing (on an HP 1009A). This process cannot be conducted in the reversed order, because the chemistry for carboxyl-terminal sequencing blocks the amino-terminus. Teflon-bound samples can also be acid-hydrolyzed for amino acid analysis and treated with proteases in situ for peptide mapping and sequencing, illustrating the versatile nature of this membrane support.
Capillary Electrophoresis
Figeys, D., van Oostveen, I., Ducret, A. and Aebersold, R. (1996) Analytical Chemistry 68, 1822-1828. Protein Identification by Capillary Zone Electrophoresis/Microelectrospray Ionization-Tandem Mass Spectrometry at the Subfemtomole Level.
The high resolution of capillary zone electrophoresis is combined with collision induced dissociation (CID) in an electrospray-ionization triple-quadrupole tandem mass spectrometer (ESI-MS/MS). About 50 fmol of trypsin-digested sample allowed proteins to be identified by database searching. Evaluating the limits of detection shows that 300 amol is needed for identifications based on ESI-MS and 600 amol for identifications from CID spectra.
Castagnola, M., Cassiano, L.M., Messana, I., Paci, M., Rossetti, D.V. and Giardina, B. (1996) Journal of Chromatography A 735, 271-281. Effect of 2,2,2-trifluoroethanol on capillary zone electrophoretic peptide separations.
Capillary electrophoresis of peptides in aqueous-organic media is improved by the addition of trifluoroethanol, frequently used to induce helical formation in peptides. Trifluoroethanol (i) increases the number of theoretical plates through viscosity and heat-dissipation effects, (ii) influences peptide ionization by lowering the dielectric constant of the media, (iii) alters sample mobilities through sequence -dependent changes in Stokes radii, (iv) improves apolar peptide separations, and (v) stabilizes the capillary coating.
Carbohydrate Structure and Analysis
Rouse, J.C. and Vath, J.E. (1996) Analytical Biochemistry 238, 82-92. On-the-Probe Sample Cleanup Strategies for Glycoprotein-Released Carbohydrates Prior to Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Spectrometry.
Small ions and detergents are used in glycosidase treatments to release protein-bound carbohydrates, but these contaminants degrade MALDI-MS signal intensities and peak resolution. Including chromatographic media with MALDI matrices reduces interference from these common contaminants, e.g., ion-exchangers for small ions and Extractigel-D for detergents. This rapid, in situ procedure improves analysis of low-pmol amounts of carbohydrates.
DNA Analysis and Sequencing
Rudd, K.E. (1996) Trends in Genetics 12, 156-157. Escherichia coli K-12 on the Internet.
Brief review describing 13 WWW Homepages and 2 anonymous FTP sites with E. coli genomic sequence data files and analysis programs. These include sites for the Genetic Stock Center (with a query -
capable database), the Encyclopedia of Genes and Metabolism, and Genome Centers in the U.S., Europe, and Japan.
Tatusov, R.L., Mushegian, A.R., Bork, P., Brown, N.P., Hayes, W.S., Borodovsky, M., Rudd, K.E. and Koonin, E.V. (1996) Current Biology 6, 279-291. Metabolism and evolution of Haemophilus influenzae deduced from a whole-genome comparison with Escherichia coli.
A glimpse at the future of genetics, where entire genomes are compared to deduce the possible functions of newly discovered genes and to derive phylogenetic relationships more meaningful than those based on physiology. H. influenzae putative protein sequences (1,703 representing 100% of the genome) were compared to E. coli sequences (3,010, about 75% of the genome), revealing 1,128 orthologous proteins, on average 59% identical. Despite this high level of protein sequence similarity, the genomes of the two organisms are remarkably different, in organization, gene order, and functionality. The entire metabolism of the two organisms could be compared and alternative metabolic pathways proposed without doing any further biochemical experiments.
Southern, E.M. (1996) Trends in Genetics 12, 110-115. DNA chips: analyzing sequence by hybridization to oligonucleotides on a large scale.
The pioneer in immobilized nucleic acid hybridization technology provides an overview of recent developments in array-sequencing methods and a persuasive argument for emphasizing development of some type of massively parallel technology for DNA sequencing over serial processes. The review emphasizes the manufacture and use of DNA chips, where large numbers of oligonucleotides with defined sequences are attached to solid supports at specific "addresses" and in a way that permits hybridization and detection. The author claims that DNA chips "could bring to sequence analysis...many of the advantages that semiconductor devices brought to computing".
Mass Spectrometry
Przybylski, M. and Glocker, M.O. (1996) Angewandte Chemie International Edition (English) 35, 806-826. Electrospray Mass Spectrometry of Biomacromolecular Complexes with Noncovalent InteractionsNew Analytical Perspectives for Supramolecular Chemistry and Molecular Recognition Processes.
Detailed practical review on the use of ESI-MS and MALDI-MS to observe and characterize enzyme-substrate, receptor-ligand, antigen -antibody, and protein-protein/nucleotide noncovalent complexes. "How-to" tips relating to sample preparation and instrumental conditions are given to increase the likelihood of successful analysis of these sample types. Many examples from "real-world" molecular biology and protein/peptide chemistry are presented.
Qin, J. and Chait, B.T. (1996) Analytical Chemistry 68, 2108-2112. Matrix-Assisted Laser Desorption Ion Trap Mass Spectrometry: Efficient Isolation and Effective Fragmentation of Peptide Ions.
A new sample excitation scheme for MALDI-MS termed "red-shifted off-resonance large-amplitude excitation" (RSORLAE) is described. Tandem MS is performed on a MALDI-ion trap instrument, allowing peptide sequencing. Ions up to about 3,500 Da can be fragmented with trapping efficiencies approaching 95%, thus dramatically improving sensitivity. (See also Qin, J. and Chait, B. T. (1996) Analytical Chemistry 68, 2102-2107, Matrix-Assisted Laser Desorption Ion Trap Mass Spectrometry: Efficient Trapping and Ejection of Ions, for details on instrumental conditions, electronics, and data analysis for RSORLAE.)
Zhao, Y., Muir, T.W., Kent, S.B.H., Tischer, E., Scardina, J.M. and Chait, B.T. (1996) Proceedings of the National Academy of Sciences (USA) 93, 4020-4024. Mapping protein-protein interactions by affinity-directed mass spectrometry.
An immunoprecipitation-mass analysis strategy to define linear epitopes is presented. The protein antigen is enzymatically digested, and a portion of the unfractionated mixture is analyzed by MALDI-MS; the remainder is then immunoprecipitated with antigen-specific antibody and mass analyzed. Comparison of the two spectra identifies candidate peptide epitopes. Only small quantities of antigen are consumed, and several enzymes can be used to further delineate epitope boundaries. Post-source decay could be used to partially assign epitope sequences.
Peptides-Chemistry and Purification
Girault, S., Chassaing, G., Blais, J.C., Brunot, A. and Bolbach, G. (1996) Analytical Chemistry 68, 2122-2126. Coupling of MALDI-TOF Mass Analysis to the Separation of Biotinylated Peptides by Magnetic Streptavidin Beads.
The well-known high-affinity interaction of biotin with avidin is exploited to mass analyze 100 fmol amounts of biotinylated peptides in complex mixtures. The biotinylated peptides are selectively adsorbed to magnetic beads conjugated with avidin. When the beads are treated with acidic MALDI matrix solutions, the peptides elute and the beads are removed magnetically. This procedure could be useful to explore peptide-protein interactions in cell extracts.
Protein Characterization and Analysis
Lottspeich, F. (editor) (1996) Electrophoresis 17, 811-966. Paper Symposium: Electrophoresis and Amino Acid Sequencing.
Comprehensive review issue devoted to electrophoresis methods for isolating sub-picomole amounts of samples for subsequent characterization by Edman and MS/MS sequencing. Contains 17 articles by expert authors representing the leading laboratories in this field.
Wheeler, C.H., Berry, S.L., Wilkins, M.R., Corbett, J.M., Ou, K., Gooley, A.A., Humphery-Smith, I., Williams, K.L. and Dunn, M.J. (1996) Electrophoresis 17, 580-587. Characterization of proteins from two-dimensional electrophoresis gels by matrix-assisted laser desorption mass spectrometry and amino acid analysis.
Coupling amino acid analysis and peptide mass fingerprinting (after proteolytic digestion) by MALDI-MS allows identification of proteins separated on two-dimensional gels, in this case human cardiac proteins.
Nakayama, H., Uchida, K., Shinkai, F., Shinoda, T., Okuyama, T., Seta, K. and Isobe, T. (1996) Journal of Chromatography A 730, 279-287. Capillary column high-performance liquid chromatographic -electrospray ionization triple-stage mass spectrometric analysis of proteins separated by two-dimensional polyacrylamide gel electrophoresis. Application to cerebellar protein mapping.
Yet another study on identifying proteins after two-dimensional gel electrophoresis. Here copper-stained proteins are excised and extracted from the gel, desalted by HPLC, concentrated, and analyzed by capillary LC-ESI-triple-stage quadrupole mass spectrometry (TSQMS)and, if necessary, subjected to proteolytic digestion. Mid -fmol sample amounts (ng quantities of gel spots) provided sequence data, which were then used to identify proteins by database searching.
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