Created: 3rd January 1999, last updated: 4th January 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, 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
Kamo M, Tsugita A. Specific cleavage of amino side chains of serine and threonine in peptides and proteins with S-ethylfluorothioacetate vapor. European Journal of Biochemistry 1998;255:162-171.
Peptides and proteins subjected to S-ethyltrifluorothioacetate vapor show cleavage on the N-terminal side of serine and threonine residues. Cleavage with this reagent, which is available commercially, is performed on protein/peptide dried in a tube that is sealed inside a larger tube containing the reagent. Incubation is performed at 50°C for 6-24 hr. Cleavage on either the N- or C-terminal side of some glycine residues also sometimes occurs, but is suppressed under anhydrous conditions. Cleaved peptides are susceptible to Edman degradation and to peptide mass fingerprinting. The method also works on proteins immobilized on PVDF membranes.
Carbohydrates and Glycoproteins
Packer NH, Harrison MJ. Glycobiology and proteomics: is mass spectrometry the Holy Grail? Electrophoresis 1998;!9:1872-1882.
A review, written for the non-mass spectrometrist, describing the utility of mass spectrometric approaches to analyzing the glycan component of glycoproteins separated by 2-D PAGE. The authors suggest that the new suite of approaches has sufficiently high throughput and sensitivity to permit investigation of the heterogeneity of glycosylation of large numbers of proteins in the context of proteomic studies.
Genes - Cloning, Sequencing and Expression
Chittum HS, Lane WS, Carlson BA, Roller PP, Lung F-DT, Lee BJ, Hatfield DL. Rabbit beta-globin is extended beyond its UGA stop codon by multiple suppressions and translational reading gaps. Biochemistry 1998;37:10866-10870.
Shows that for at least one eukaryotic gene, in vivo translation can skip the first in-frame stop codon by "hopping" over this codon, or by using suppressor tRNAs to read it as an amino acid. The first of these mechanisms, in which a translational reading gap occurs, has previously been observed only in prokaryotes. The phenomenon is documented here in the case of rabbit beta-globin. Seven different proteolytic peptides from the read-through protein were sequenced by quadrupole tandem mass spectrometry in an ion trap and shown to be derived from the same DNA sequence, which spans the stop codon. This phenomenon, although unlikely to be common, should be born in mind by all those translating ORFs in genome sequences. The paper also utilizes a program called FuzzyIons that facilitates manual, de novo sequencing of peptides using MS/MS data. The program is web-based, and uses a graphical, bi-directional amino acid "ruler" against which to visually measure off intervals between fragment ions. Amino acids on the ruler that align with fragment ions in the spectrum are automatically bolded.
Salas-Solano O, Carrilho E, Kotler L, Miller AW, Goetzinger W, Sosic Z, Kerger BL. Routine DNA sequencing of 1000 bases in less than one hour by capillary electrophoresis with replaceable linear polyacrylamide solutions. Analytical Chemistry 1998;70:3996-4003.
Multicapillary instruments capable of running 96 or more samples simultaneously are now being commercialized. This paper describes the optimization of a variety of conditions for high through-put sequencing using such instruments, including the composition of linear acrylamide polymers used as the matrix for electrophoresis, the electrophoretic conditions employed, the use of BigDye-labeled primers, and automated base-calling. With these optimized procedures, runs of 1000 bases were achieved in less than 55 minutes at an accuracy greater than 99% for single-stranded templates.
Mass Spectrometry
Gevaert K, Demol H, Sklyarova T, Vandekerckhove J, Houthaeve T. A peptide concentration and purification method for protein characterization in the subpicomole range using matrix assisted laser desorption/ionization-postsource decay (MALDI-PSD) sequencing. Electrophoresis 1998;19:909-917.
Reverse phase chromatographic beads are used to concentrate peptides from very dilute solutions by evaporating peptide-bead suspensions to dryness under vacuum. The peptides bind hydrophobically to the beads, and may subsequently be eluted in a small voume of MALDI matrix dissolved in an aqueous/organic solvent, and transferred to a MALDI target for mass analysis (the beads may be transferred onto the target along with the liquid). By washing the peptide-bound beads before elution, samples that contain high concentrations of some substances that interfere with MALDI, such as buffer salts, urea, guanidine, and glycerol, can be cleaned up. Storage of peptides in bead-bound form is also successful in preventing sample loss, even with samples in low femtomole amounts.
Figeys D, Aebersold R. Nanoflow solvent gradient delivery from a microfabricated device for protein identifications by electrospray ionization mass spectrometry. Analytical Chemistry 1998;70:3721-3727 and Figeys D, Gygi SP, McKinnon G, Aebersold R. An integrated microfluidics-tandem mass spectrometry system for automated protein analysis. Analytical Chemistry 1998;70:3728-3734.
A pair of papers describing the construction and utilization of a microfabricated device for the generation and delivery of solvent gradients at nanoliter per minute flow rates. The device is etched in glass. Solvent gradients are generated by differential electroosmotic pumping of an aqueous and an organic solvent present in different reservoirs on the device. This is done by varying the potential applied to the respective reservoirs. Peptides generated by proteolytic digestion of gel-separated proteins are fractionated by using this solvent gradient device in conjunction with a small reverse phase cartridge and then analyzed by an electrospray ion trap mass spectrometer. This system is used for the sequential, automated analysis of proteins. Quantities as low as 1 fmol of protein are detected.
Zhang W, Niu S, Chait BT. Exploring infrared wavelength matrix-assisted laser desorption/ionization of proteins with delayed-extraction time-of-flight mass spectrometry. Journal of the American Chemical Society 1998;9:879-884.
Primarily a study of the application of delayed extraction to IR-MALDI, this paper also includes a systematic comparison of UV- and IR-MALDI that will be of interest to those practicing the former technique. Photochemical matrix adduction, which is a feature of UV-MALDI is absent in IR-MALDI, and protein ions produced by IR-MALDI are observed to be less internally excited, showing less fragmentation, more Na+ replacement or unspecified non-covalent adduction, and more adduction of heme to apomyoglobin. IR-MALDI therefore seems to be a softer ionizing technique that may become generally useful for the study of large proteins.
Peptides - Synthesis
Tam JP, Lu Y-A. A biomimetic strategy in the synthesis and fragmentation of cyclic protein. Protein Science 1998;7:1583-1592.
A description of the synthesis of two naturally occurring plant peptides, circulin B and cyclopsychotride. These are both cyclic peptides with 31 amino acids, and have three disulfide bonds of connectivity 1-4, 2-5, and 3-6. The cyclization reaction uses a simple and efficient scheme which begins with a linear peptide that has a thioester bond at the C-terminus. This forms an alpha-amino lactone with the alpha-amino group at the N-terminus, a reaction that proceeds through a series of reversible thiol-thiolactone exchanges involving the internal cysteine side chains (the so-called "thia zip reaction"). Finally, the alpha-amino thiolactone undergoes an irreversible S-->N acyl migration to form the cyclic peptide. The disulphides are formed in a reaction sequence that has only two steps, unlike more conventional schemes that would require multi-tiered protecting groups. Confirmation of the disulfide connections was performed by fragmenting the peptides by partial acid hydrolysis.
Kaplan BE, Hefta LJ, Blake RC II, Swiderek KM, Shively JE. Solid-phase synthesis and characterization of carcinoembryonic antigen (CEA) domains. Journal of Peptide Research 1998;52:249-260.
Description of the high efficiency synthesis of four long peptides (84-184 residues) using the Fmoc chemistry. The petides corresponded to immunoglobulin-like domains of carcinoembryonic antigen. An automated synthesizer built in-house was employed. Elevated temperatures were employed to promote the coupling and deprotection reactions. A DMSO/DCM/NMP solvent mixture was used. This was gradually distilled away by bubbling gas through the vessel during coupling. This concentrated the reactants and progressively changed the composition of the solvent so as to become optimal at some stage during the reaction. Peptides were synthesized with a His6 tail at the N-terminus and purified on a Ni-NTA chelate column to remove the expected large amounts of impurities.
Proteins - Purification and Characterization
Molloy MP, Herbert BR, Walsh BJ, Tyler MI, Traini M, Sanchez J-C, Hochstrasser DF, Williams KL, Gooley AA. Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 1998;19:837-844.
Describes a procedure for fractionation of E. coli lysates by differential solubilization. The procedure is designed for use with 2-D PAGE employing immobilized pH gradients. It consists of three successive extractions. The first uses Tris base, and yields mainly cytosolic proteins. The insoluble material remaining is then treated with a cocktail containing urea, CHAPS and DTT. This liberates most of the proteins present. However, the insoluble material from this step is then treated with urea, thiourea, tributyl phosphine and zwitterionic surfactants, and yields a fraction enriched with membrane proteins. By running the three extracts on different gels, protein identification and gel matching is facilitated because the complexity of the patterns is reduced.