Created: 28th February 1999, last updated: 7th April 1999, © 1999 ABRF
This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, HHMI/University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9050; Tel: (214) 648-5051; Fax: (214) 648-9477; email: slaugh01@utsw.swmed.edu; or to any member of the editorial board. Article summaries reflect the reviewers' opinions and not necessarily those of the Association.
Kaiser R, Metzka L. Enhancement of cyanogen bromide cleavage yields for methionyl-serine and methionyl-threonine peptide bonds. Anal Biochem 1999;266:1-8.
Cyanogen bromide (CNBr) is a commonly used protein fragmentation reagent that cleaves on the C-terminal side of Met residues. Formic acid (70%) is the solvent normally employed, partly because it is a good protein solvent; however, it provides low cleavage efficiency at Met-Ser and Met-Thr bonds. In the course of this study of the side products of CNBr treatment, cleavage yields at these bonds were found to be doubled by increasing the concentration of water during the reaction. This is achieved by using lower concentrations of formic acid or by using acidic aqueous solvents (eg, 7 M urea/0.1 N HCl) that have protein solubilizing properties comparable to formic acid.
Wright SK, Viola RE. Evaluation of methods for the quantitation of cysteines in proteins. Anal Biochem 1998;265:8-14.
The standard method for cysteine quantitation, titration with DTNB (Ellman's assay), is demonstrated to be reliable at thiol concentrations as low as 0.3 µM using carefully controlled conditions. The rate of reaction varies with the solvent accessibility of the cysteines; 10-fold greater sensitivity is achieved with an assay that relies on the ability of cysteines on proteins to reactivate papain that has previously been inactivated through reaction of the thiol in its catalytic center with methyl methanethiosulfonate. However, even better sensitivity is observed with the ThioGlo reagent, a fluorogenic maleimide-derivatized naphthopyranone, with which protein thiol concentrations as low as 10 nM can be determined.
Kheterpal I, Mathies RA. Capillary array electrophoresis DNA sequencing. Anal Chem 1999;71:31A-37A.
This review of contemporary technology for high-throughput DNA sequencing using arrays of multiple capillaries includes a comparison of the various optical detection systems that have been used with capillary arrays and an account of recent developments in fluorescent labels and sieving matrices. Although capillary arrays allow major increases in sequencing capacity and speed, these systems do have limitations. For example, as the number of capillaries in the array is increased, manufacturing becomes more difficult, and limitations in injection efficiency are encountered. However, microfabricated array systems are helping to solve these problems, and separations with microchip DNA sequencing devices are approaching the quality obtained with conventional capillaries.
Winzeler EA, Richards DR, Conway AR, et al. Direct allelic variation scanning of the yeast genome. Science 1998;281:1194-1197.
In this application of high-density oligonucleotide arrays, total genomic DNA from two different strains of yeast was hybridized to arrays that covered 22% of the nonrepetitive regions of the yeast genome, and differences in hybridization were analyzed. On the arrays, 3714 probes were identified as reliably distinguishing the two strains, enabling their use as allelic markers. The average spacing between the markers was calculated to be 3.5 kb, and they constituted 4.7% of the estimated total variation between the strains. The markers were then used to map a drug-resistance locus and four other loci simultaneously with a resolution of 11 to 64 kb. This method offers substantial advantages over other methods for scanning or scoring markers in that it does not depend on allele-specific polymerase chain reactions, amplification steps, gels, or enzymatic manipulations.
Erdjument-Bromage H, Lui M, Lacomis L, et al. Examination of micro-tip reversed-phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis. J Chromatogr A 1998;826:167-181.
Eppendorf gel loading tips filled with reverse-phase chromatographic media are used for sample clean-up and concentration in experiments to identify proteins by peptide mass fingerprinting. Peptide recoveries of 65% to 70% are demonstrated by radioactive tracer methods. Optimal elution of peptides is observed with 0.1% formic acid/70% water/30% acetonitrile. Zwittergent detergent is suggested for improving recovery of peptides from digests of small quantities of proteins but requires sufficient reverse-phase medium to bind the detergent and the peptides. Stepwise elution of peptides with different concentrations of acetonitrile results in partial fractionation of peptides and larger numbers of peptide signals.
Neubauer G, Mann M. Mapping of phosphorylation sites of gel-isolated proteins by nanoelectrospray tandem mass spectrometry: potentials and limitations. Anal Chem 1999;71:235-242.
Methods used in the Heidelberg laboratory for identifying phosphorylation sites are described. Electrophoretically purified proteins are digested in gel with trypsin or Lys-C and the digests desalted in capillaries containing a reverse-phase column-packing material. A packing material designed for purification of oligonucleotides is employed to minimize loss of small hydrophilic peptides, and elution conditions are adjusted for compatibility with electrospray ionization by using basic pH. Phosphopeptides are introduced into a triple quadrupole mass spectrometer by nanoflow methods and detected by scanning for precursors of m/z 79 (phosphate) in the negative ion mode. The success of this strategy is obviously influenced by the occupancy of the phosphorylation site or sites, but it also depends on finding an enzyme that yields a peptide with high efficiency in the right size range to be detected by the precursor ion scan. Initial experiments may be needed to determine which enzyme works best in a particular case.
Hemmer B, Pinilla C, Appel J, Pascal J, Houghten R, Martin R. The use of soluble synthetic peptide combinatorial libraries to determine antigen recognition of T cells. J Pept Res 1998;52:338-345.
A study of antigen recognition by the T-cell receptor using synthetic peptide combinatorial libraries. Positional scanning was employed to investigate the requirements of T-cell clones for binding to peptides presented on class I MHC molecules. The results indicate that T-cell clones can recognize a large number of different peptide sequences and that individual T-cell receptors recognize peptides in a highly flexible manner. Some peptides may even show better recognition than the original peptide used to stimulate growth of the T-cell clone in culture. High-affinity ligands with sequences derived from microbial sources may also be identified using data derived from the positional scanning approach. The importance of this is that microbial sequences that cross-react with self-epitopes constitute agents for the potential induction of autoimmune disease.
Appel JR, Campbell GD, Buencamino J, Houghten RA, Pinilla C. Characterization of antigen-antibody interactions using single substitution analogs and mixture-based synthetic combinatorial libraries. J Pept Res 1998;52:346-355.
Two approaches are compared for determining the sequence requirements for binding of a monoclonal antipeptide antibody to its peptide antigen. The first approach uses single substitution analogs of the peptide, and the second uses positional scanning synthetic combinatorial libraries. The two methods gave concordant results, validating the combinatorial approach. The latter has the advantage that one library can be screened against any monoclonal antibody without prior knowledge of the antibody's specificity. Furthermore, a single screen permits the identification of the residues that are critical for binding.
Kameshita I, Ischida A, Fujisawa H. Analysis of protein-protein interaction by two-dimensional affinity electrophoresis. Anal Biochem 1998;262:90-92.
An electrophoretic method is presented for detecting protein-protein interactions in which the protein or protein mixture of interest is first subjected to native polyacrylamide gel electrophoresis (PAGE) through a gel into which the potential interacting protein has been incorporated before gel polymerization. Protein-protein interaction is detected as a retardation of electrophoretic mobility of the protein or proteins of interest relative to samples subjected to electrophoresis in the absence of putative ligand. The method is shown to detect interactions of proteins with calmodulin in the presence of Ca2+ ions.
Jaffe H, Veeranna, Pant HC. Characterization of serine and threonine phosphorylation sites in beta-elimination/ethanethiol addition-modified proteins by electrospray tandem mass spectrometry and database searching. Biochemistry 1998;37:16211-16224.
A method is described for characterizing serine and threonine phosphorylation sites that involves chemical modification. Sites on the intact phosphoprotein are subjected to beta-elimination and ethanethiol addition to convert phosphoserine to S-ethylcysteine and phosphothreonine to beta-methyl-S-ethylcysteine. The protein is then digested and analyzed by microbore liquid chromatography and electrospray ionization. An ion trap mass spectrometer is used to produce MS/MS spectra. The uninterpreted spectra are subjected to database searching using the SEQUEST program. The chemical modification approach was found to be advantageous because it produced diasteriomers that eluted at different retention times, increasing the chances of target peptide identification; increased the hydrophobicity of peptides and hence increased their retention times; facilitated positive ion production; and increased susceptibility to digestion with trypsin by converting negatively charged phosphorylated residues to neutral residues.