Created: 19^th June 1998, last updated: 19^th June 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, UT Southwestern, 5323 Henry Hines Blvd, Dallas, TX 75235-9050. Tel: (214) 648-5051, Fax: (214) 648-9477, E-mail: slaugh01@utsw.swmed.edu or to any member of the Editorial Board. Article summaries reflect the reviewer's opinions and not necessarily those of the Association.

Amino Acid Composition and Sequence Analysis

Gheorghe MT, Jornvall H, Bergman T. Optimized alcoholic deacetylation of N-acetyl-blocked polypeptides for subsequent Edman degradation. Analytical Biochemistry 1997;254:119-125.
Describes a chemical method for deblocking peptides and proteins that are Na-acetylated for subsequent Edman degradation. The method involves incubation with trifluoroacetic acid in methanol at elevated temperature for 2-3 days. Yields of residues from the deblocked sequence are often high compared to the background yields of residues from internal peptide bond cleavage (up to 10:1 for peptides and >2:1 for proteins).

Thannhauser TW, Sherwood RW, Scheraga HA. Determination of the cysteine and cystine content of proteins by amino acid analysis: application to the characterization of disulphide-coupled folding intermediates. Journal of Protein Chemistry 1998;17:37-43.
Cysteine residues are first blocked with 2-aminoethyl methanethiosulfonate (AEMTS), which converts all free thiol groups to mixed disulfides with 2-aminoethanethiol (AET). The blocked protein is then oxidized with performic acid, which converts the AET blocking groups to taurine, and all cysteine residues, including those involved in disulphide bonds, to cysteic acid. Both these derivatives are stable to subsequent hydrolysis, and are recovered quantitatively for amino acid analysis. The reaction with AETMS is fast and specific enough to make this method useful for characterizing disulphide-coupled folding intermediates.

Hardin SC, Wolniak SM. Low-voltage separation of phosphoamino acids by silica gel thin-layer electrophoresis in a DNA electrophoresis cell. BioTechniques 1998;24:344-346.
A detailed description of a simple, rapid method that resolves phospho-Ser, -Thr, and -Tyr. The method is one that most laboratories are equipped to perform, avoiding the use of expensive power supplies and temperature-regulating equipment. It involves 1-D, low voltage electrophoresis on pre-formed, silica-gel plates in a DNA electrophoresis cell.

Carbohydrates and Glycoproteins

Cancilla MT, Penn SG, Lebrilla CB. Alkaline degradation of oligosaccharides coupled with matrix-assisted desorption/ionization Fourier transform mass spectrometry: a method for sequencing oligosaccharides. Analytical Chemistry 1998;70:663-672
Describes a new technique for determining the sequence and linkage of underivatized oligosaccharides. The technique uses alkaline degradation, also known as the 'peeling reaction', a chemical degradation technique that cleaves the glycosidic bond only at the reducing end, yielding a new reducing end. The reaction products are sampled and monitored by MALDI-FTMS to determine the oligosaccharide sequence. Linkage information is provided using cross-ring cleavage produced by fragmentation of the new reducing ends in the mass spectrometer. This fragmentation can be performed either in the MALDI source or in tandem MS/MS mode.

Genes - Cloning, Sequencing and Expression

Duttweiler HM, Gross DS. Bacterial growth medium that significantly increases the yield of recombinant plasmid. BioTechniques 1998;24:438-444.
Describes a growth medium, called H15, for culture of E. coli to isolate plasmid DNA. The medium supports 30-fold higher cell densities, and correspondingly elevated yields of plasmid, than conventional rich media. This phenomenon is independent of E. coli strain, DNA insert size, and copy number. The medium is also very economical: as much as 6 mg of plasmid can be harvested per dollar of medium.

Mass Spectrometry

Williams ER. Tandem FTMS of large biomolecules. Analytical Chemistry 1998;70:179A-185A
Fourier transform mass spectrometry (FTMS) has remarkable capabilities. This review, which is suitable for non-experts, discusses principles and applications of tandem Fourier transform mass spectrometry coupled with electrospray ionization. Cited examples include isotopic resolution of chondroitinase II (112 kDa), detection of hemoglobin a and b chains from a single erythrocyte (450 amol), and the fragmentation of intact carbonic anhydrase (259 amino acids) to obtain spacings for 23 amino acids.

Arnott D, O'Connell KL, King KL, Stults JT. An integrated approach to proteome analysis: identification of proteins associated with cardiac hypertrophy. Analytical Biochemistry 1998;258:1-18.
An example of protein identification from 2-D gels by a combination of MALDI-TOF peptide mass fingerprinting and ion trap peptide sequence analysis. In this study, low levels of SDS are incorporated into the digestion buffer during in-gel protein digestion, with a substantial improvement in the recovery of peptides for mass analysis.

Reiber DC, Grover TA, Brown RS. Identifying proteins using matrix-assisted laser desorption/ionization in-source fragmentation data combined with database searching. Analytical Chemistry 1998;70:673-683
Metastable ion decay can be observed in the MALDI ion source of linear TOF mass spectrometers equipped with delayed extraction capability, and can provide sequence information for peptides. C- and N-terminal sequence information can be obtained for even moderately sized peptides (5000 Da). For larger (>5000 Da) peptides and proteins, a unique cn ion series predominates and permits C-terminal sequencing. This paper demonstrates sequence acquisition from large CNBr peptides and intact proteins that can be used for database searching. The method is of interest to those who would like to use tandem MS for database searching but who are limited to conventional TOF instrumentation.

Worrall TA, Cotter RJ, Woods AS. Purification of contaminated peptides and proteins on synthetic membrane surfaces for matrix-assisted laser desorption/ionization mass spectrometry. Analytical Chemistry 1998;750-756.
A method is described for removing salt, detergent and glycerol contaminants from peptides that are to be analysed by MALDI. Such contaminants prevent effective MALDI from conventional steel surfaces. Contaminated peptides are applied to polyethylene, polypropylene, C8 or C18 membranes, and washed before addition of matrix. Polyethylene and polypropylene surfaces also provide better resolution than stainless steel.

Nucleic Acids - Synthesis

Marshall WS, Boymel JL. Oligonucleotide synthesis as a tool in drug discovery research. Drug Discovery Today 1998;3:34-42.
Describes how Amgen, Inc. (Boulder, CO) set up a highly automated, high-throughput oligonucleotide synthesis facility. The facility uses PE/ABD DNA synthesizers to make up to 300 oligonucleotides per day. A variety of automated workstations perform cleavage and deprotection, purification, quantitation, dispensing, and MALDI-TOF analysis. The entire system is controlled through a 4-D relational database, which handles all sample tracking and data transfer. This article is of interest as a description of the steps involved in the design and implementation of a highly automated laboratory. Furthermore, the system was implemented in only one year.

Peptides - Synthesis and Purification

Camarero JA, Cotton GJ, Adeva A, Muir TW. Chemical ligation of unprotected peptides directly from a solid support. Journal of Peptide Research 1998;51:303-316.
Describes a simple strategy for conducting native intramolecular (cyclization) and intermolecular peptide ligations that involves only solid-phase manipulations. The elimination of solution-phase steps prior to ligation results in reduced handling losses and enhanced efficiency of the chemical procedure. A fully de-protected peptide that is still attached to the solid support via an a-thioester linkage is first produced. The immobilized peptide a-thioester then participates in a ligation reaction with a free N-terminal cysteine residue on the same peptide molecule (cyclization) or a different molecule (intermolecular ligation) to produce a native (normal) peptide bond. After the reaction, the ligation product is simply removed by filtration.

Proteins - Purification and Characterization

Paper symposium: affinity capillary electrophoresis. Heegaard NHH, (Editor). Electrophoresis 1998;19(3).
A collection of 15 papers describing studies in which reversible intermolecular interactions taking place during electrophoresis affect the selectivity of the separation process. Such effects, combined with the high resolution of capillary CE, provide detailed information about molecular interactions. The papers cover a broad range of topics, including immunochemistry, receptor chemistry, studies of chiral compounds, and lectin-glycoconjugate interactions.

Hubbard SJ. The structural aspects of limited proteolysis of native proteins. Biochimica et Biophysica Acta 1998;1382:191-206.
A review of limited proteolysis as a tool for investigating protein structure. Structural determinants of cleavage sites, attempts to predict the location of sites from structure and sequence, and examples illustrating the use of limited proteolysis are discussed.

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