Created: 8th September 1998, last updated: 9th September 1998, © 1998 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, U.T. Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas TX 75235-9050. Tel: (214) 648-505, Fax: (214) 648-9477, E-mail; 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.
Amino Acid Composition and Sequence Analysis
Hsi K-L, O’Neill SA, DuPont DR, Yuan P-M. Visualization of proteins by modification of lysines, cysteines, and phosphorylated serines facilitates sample preparation for microsequencing. Analytical Chemistry 1998;258:38-47.
A method is devised for detecting proteins following SDS-PAGE that uses fluorescent or chromogenic probes as tags for certain amino acids. Prior to electrophoresis, proteins are derivatized with dansyl/dabsyl chloride to label lysines, or with iodoacetamidofluorescein (I-15) to label cysteines. I-15 may also be used to tag phosphoserines if ethanedithiol is first employed for b-elimination of the phosphate. Sensitivities of protein detection are comparable to those observed with Coomassie blue staining, but the labelling methods eliminate the need for fixing, staining and destaining. The labels can also be used for selective isolation of lysine-, cysteine- or phosphoserine-containing peptides after enzymatic digestion of the labelled protein.
Hardeman K, Samyn B, van der Eycken J, van Beeumen J. An improved chemical approach toward the C-terminal sequence analysis of proteins containing all natural amino acids. Protein Science 1998;7:1593-1602.
A revisitation of chemical methods for the serial degradation of peptides from the C-terminus. Conditions are identified that permit the derivatization of all 20 standard amino acids to the corresponding thiohydantoins. These conditions involve activation by acetyl chloride and derivatization with ammonium thiocyanate. C-terminal prolines were derivatized with 30-60% yield. Mass spectrometric data indicate that proline can form an oxazolonium ion that reacts with thiocyanate in the same general way as other amino acids.
Carbohydrates and Glycoproteins
Sheeley DM, Reinhold VN. Structural characterization of carbohydrate sequence, linkage, and branching in a quadrupole ion trap mass spectrometer: neutral oligosaccharides and N-linked glycans. Analytical Chemistry 1998;70:3053-3059.
Demonstrates the use of a commercial ion trap mass spectrometer to obtain linear sequence, linkage and branching information for several oligosaccharides and N-linked glycans. The multistage MS/MS (MSn) capability of the ion trap permitted the assignment of linkages that cannot be made with triple quadrupole mass spectrometers, and the results foreshadow the development of strategies for the complete structural characterization of carbohydrates using a single instrument.
Genes - Cloning, Sequencing and Expression
Berkenkamp S, Kirpekar F, Hillenkamp F. Infrared MALDI mass spectrometry of large nucleic acids. Science 1998;281:260-262.
Describes progress in the development of methods for high accuracy mass measurement of DNA fragments obtained by restriction enzyme digestion. An infrared laser is used, in conjunction with liquid glycerol as the MALDI matrix. The conditions permit mass measurement of fragments up to about 700 kDa (2,200 nucleotides) with mass accuracies of 1% or better and resolution values around 50. By contrast, electrophoretic methods are of limited resolution and provide accuracies of only 5-10%. The signals typically represent a composite of the two complementary DNA single strands. Large RNA can also be measured. Sensitivities in the low femtomole range are routine, although a spectrum of a 300 amol sample is also demonstrated.
Mass Spectrometry
Lehmann WD. Single series peptide fragment ion spectra generated by two-stage collision-induced dissociation in a triple quadrupole. Journal of the American Society for Mass Spectrometry 1998;9:606-611.
Collision-induced dissociation (CID) of peptides in the source region of a triple quadrupole mass spectrometer, together with precursor ion scanning or neutral loss scanning, is used to provide spectra showing signals of predominantly a single ion series, such as the b or y series. Collisional dissociation induced by adjustment of the skimmer voltage, combined with scanning for neutral loss of 28, generates spectra showing b ions. Skimmer CID of tryptic peptides, combined with scanning for precursors of the y1 fragment corresponding to the C-terminal arginine (m/z 147), or the y1 fragment corresponding to the C-terminal lysine (m/z 175), generates spectra showing the y series. Sequence information can be easily extracted from these simplified spectra because the scan mode defines the type of fragments observed.
Zhang X, Herring CJ, Romano PR, Szczepanowska J, Brzeska H, Hinnebusch AG, Qin J. Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis. Analytical Chemistry 1998;70:2050-2059.
A streamlined and sensitive method for identifying phosphorylation sites. The phosphoprotein is subjected to SDS-PAGE, subjected to in-gel digestion with trypsin, and the resulting peptides subjected to MALDI-TOF before and after digestion with a phosphatase to identify phosphopeptides. Peptides are then subjected to on-line LC/MS/MS in an ion-trap mass spectrometer to identify the precise phosphorylation sites. Using this scheme, the authors have assigned 14 phosphorylation sites. Experiments using a gel slice containing as little as 3 pmol of protein have proved sufficient for successful analysis.
Nucleic Acids - Synthesis
Focus on Oligonucleotides. McLuckey SA, (Editor). Journal of the American Society for Mass Spectrometry 1998;9:659-691.
A collection of four papers of interest to those wishing to understand current progress and issues in the development of mass spectrometric methods for the characterization of oligonucleotides. The contributions include, firstly, methods for confirming sites of addition, and structural features, of labels incorporated by phosphodiester bonds into the oligonucleotide backbone; secondly, a method that reduces the effects of sodium adducts and diminishes the fragmentation of long oligonucleotides during MALDI; thirdly, methods for determining the site(s) at which carcinogens form adducts to bases; and, fourthly, an evaluation of methods that permit the characterization of mixtures of DNA arising by replication of oligonucleotides damaged by uv light.
Peptides - Synthesis
Miller C, Rivier J. Analysis of synthetic peptides by capillary zone electrophoresis in organic/aqueous buffers. Journal of Peptide Research 1998;51:444-451.
Describes mobile phase conditions for assessment of the purity of synthetic peptides by CZE. Triethylammonium phosphate is employed as a modifyer, and methanol, acetonitrile and isopropanol are tested at various concentrations with significant enhancements of separation efficiency compared to a standard aqueous phosphate buffer.
Jensen KJ, Alsina J, Songster MF, Vágner J, Albericio F, Barany G. Backbone amide linker (BAL) strategy for solid-phase synthesis of C-terminally modified and cyclic peptides. Journal of the American Chemical Society 1998;120:5441-5452.
Presents a new, general method for the solid-phase synthesis of peptides in which the carboxy termini are to be modified to other functionalities. Examples described include alcohols, N,N-dialkylamides, aldehydes, esters, and head-to-tail cyclic peptides. The method involves attachment of the initial residue to the polymeric support via a "handle", a bifunctional linker, at one end of which is a group that acts like a smoothly cleavable protecting group, and at the other end of which is a group that permits coupling to the previously functionalized support. The handle used in this case is a novel Backbone Amide Linker (BAL), in which the growing peptide is anchored through a backbone amide nitrogen. This approach allows considerable flexibility in the management of the terminal functionality desired, and avoids often complicated, post-synthetic, solution-phase manipulations.
Proteins — Purification and Characterization
Yan JX, Packer NH, Gooley AA, Williams KL. Protein phosphorylation: technologies for the identification of phosphoamino acids. Journal of Chromatography A 1998;808:23-41.
A concise but broad-based methodological review of protein phosphorylation. Lists the known, naturally occurring phosphoamino acids, and phophorylation sequences that are recognized by various kinases. Summarises methods for detecting phosphate moieties on proteins, including radiolabelling, use of antibodies specific for different phosphoamino acids, and fluorescence labelling. Describes methods for protein hydrolysis, and for identifying phosphoamino acids, including TLC, electrophoresis, HPLC, and mass spectrometry. Describes the b-elimination reactions undergone by Ser(P) and Thr(P), and indicates methods for locating the phosphorylation sites in proteins by Edman degradation and mass spectrometry.