Created: 1st September 2000, last updated: 30th October 2000, © 2000 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 A. Slaughter, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794; Tel: (901) 495-4844; Fax: (901) 495-2945; email: clive.slaughter@stjude.org; or to any member of the editorial board. Article summaries reflect the reviewers' opinions and not necessarily those of the Association.
Liu S, Ren H, Gao Q, Roach DJ, Loder RT, Armstrong TM, Mao Q, Blaga I, Barker DL, Jovanovich SB. Automated parallel DNA sequencing on multiple channel microchips. Proc Natl Acad Sci USA 2000;97:5369-5374.
Automated sequencing in matrix-filled channels on a 16-channel microchip is described. Samples are loaded with an 8-tip pipettor, and high voltage is then applied to reservoirs in a programmed sequence to inject and fractionate the samples. An integrated, four-color, scanning, confocal fluorescence detector is used to acquire the results. The method yields from each channel the sequence of at least 450 bases, and as many as 543 bases in ideal cases, in just 15 minutes. Analyses are complete within 18 minutes. In addition to such high speed, DNA sequencing on microchips inherently produces signals of uniform intensity for templates of different size and tolerates high template concentration.
Zhang Z, McElvain JS. De novo peptide sequencing by two-dimensional fragment correlation mass spectrometry. Anal Chem 2000;72:2337-2350.
A sequence interpretation protocol is described that exploits the ready availability of granddaughter ion (MS3) spectra from ion-trapping mass spectrometers (quadrupole ion trap and ion cyclotron instruments). The interpretation of daughter ion (MS2) spectra is complicated by the presence of both N-terminal (b ion) and C-terminal (y ion) fragments. However, isolation and fragmentation of a particular daughter ion during the acquisition of an MS3 spectrum yields mainly granddaughter ions belonging to the same series (b or y) as the daughter. Unfortunately, MS3 spectra also contain internal fragments that are typically lacking in MS2 spectra. These may be eliminated from the analysis by considering only those signals that are shared by both MS2 and MS3 spectra. A spectrum that contains only such shared signals is called an intersection spectrum. A computational method for sequence interpretation is described that is based on two-dimensional diagrams in which correlation spectra are plotted against MS2 spectra (two-dimensional fragment correlation spectra). The method is amenable to automated sequence assignment.
Rostom AA, Fucini P, Benjamin DR, Juenemann R, Nierhaus KH, Dobson CM, Robinson CV. Detection and selective dissociation of intact ribosomes in a mass spectrometer. Proc Natl Acad Sci USA 2000;97:5185-5190.
Ions corresponding to intact ribosomes are demonstrated to be produced by electrospray in a hybrid quadrupole-time-of-flight mass spectrometer. The quadrupole is used in the radiofrequency-only mode as a wide-bandpass filter, and pressures in the source and quadrupole regions are maintained at sufficiently high levels to produce collisional cooling to facilitate the transmission of very-high-mass ions. Mass measurements are made using the time-of-flight stage. Maintaining high Mg2+ concentrations in ribosome solutions and ultraviolet-induced cross-linking of ribosomal proteins to the rRNA increased the stability of intact ribosomes. Lowering the Mg2+ concentration resulted in dissociation into 30S and 50S subunits. Resolution of charge states in the spectrum of the 30S subunit allowed the mass to be determined as 852,187 ± 3918 Da, a value within 0.6% of the calculated mass. Further dissociation into smaller subcomplexes and then individual subunits were induced by increasingly energetic gas-phase collisions. The results were in accord with known interactions within the particle. This study demonstrates techniques by which mass spectrometry can be used to characterize high-molecular-weight macromolecular assemblies.
Shevchenko A, Loboda A, Shevchenko A, Ens W, Standing KG. MALDI quadrupole time-of-flight mass spectrometry: a powerful tool for proteomic research. Anal Chem 2000;72:2132-2141.
An orthogonal quadrupole-time-of-flight mass spectrometer equipped with a matrix-assisted laser desorption and ionization (MALDI) source in place of the more usual electrospray source is used for protein identification. This instrument configuration permits identification either by peptide mass fingerprinting of protein digests or by spectral analysis of fragment ions produced from individual precursor ions in a collision cell. Both kinds of spectra can be acquired from a single sample in the same experiment. The use of collision-induced dissociation to yield sequence information circumvents the low product ion yields and problematic interpretation of product ion spectra acquired by the postsource decay technique. Collisional damping is used to cool the ions produced by MALDI before they enter the quadrupole. The time-of-flight measurement is thus decoupled from the MALDI process and provides high mass accuracy for both precursor and product ions, high resolution, simple selection of precursor ions, precise tuning of collision energies, and a simplified calibration procedure.
Egelhofer V, Büssow K, Luebbert C, Lehrach H, Nordhoff E. Improvements in protein identification by MALDI-TOF-MS peptide mapping. Anal Chem 2000;72: 2741-2750.
A strategy for interrogating databases with peptide mass fingerprint data is described in which correction is made for systematic errors in mass measurements by using information contained in the databases. A search is initially performed allowing high values for maximum allowable mass deviation, and candidate proteins are identified. For each candidate protein, deviations are used to calculate correction factors. After applying correction, the number of matched mass values is recalculated using more stringent values for allowable mass deviation. Authentic candidates are distinguished from spurious ones by the magnitude of the improvement in the number of matches obtained. A program that performs these operations automatically has been made available for free downloading via the Internet (http://molgen.mpg.de).
Cancilla MT, Gaucher SP, Desaire H, Leary JA. Combined partial acid hydrolysis and electrospray ionization-mass spectrometry for structural determination of oligosaccharides. Anal Chem 2000;72:2901-2907.
A general method for acid-catalyzed cleavage of oligosaccharides that is compatible with electrospray ionization is described. In place of traditional methods, which require tedious sample purification prior to electrospray ionization, a cation exchange resin is employed that can be removed by simple filtration. Both hexose-containing and N-acetylhexosamine-containing oligosaccharides can be treated under the same conditions to yield a ladder of partial hydrolysis products which can then be analyzed directly in ion trap or ion cyclotron mass spectrometers to provide sequence information. Minimal degradation of monosaccharide residues or deacetylation of N-acetylhexosamines is encountered. The hydrolysis products contain reducing ends that can be derivatized with diethylaminetriamine and then coordinated with zinc to yield species that fragment during collisional dissociation in a manner that provides linkage information. These derivatives are further used to determine the stereochemistry of monosaccharides and the anomeric configuration of glycosidic bonds within disaccharides that are released during acid hydrolysis.
Howe J, Quibell M, Johnson T. A new generation of reversible backbone-amide protection for the solid phase synthesis of difficult sequences. Tetrahedron Letters 2000;41:3997-4001.
Sequences in which poor rates of deprotection and coupling reactions are encountered during solid-phase peptide synthesis are believed to be due to association between peptide chains stabilized by hydrogen bonds involving backbone amides. Judicial use of N-(2-hydroxy-4-methoxybenzyl) (Hmb) backbone protection inhibits the formation of such aggregated secondary structures and provides improvements in the quality of crude peptide products. This article builds on the success of the Hmb group by incorporating new design elements into a second generation of amide bond-protecting groups. Specifically, the N-(3-methylsulfinyl-4-methoxy-6-hydroxybenzyl) (SiMB) group is shown to give improved coupling kinetics relative to the Hmb group.
Locke S, Figeys D. Techniques for optimization of proteomic strategies based on head column stacking capillary electrophoresis. Anal Chem 2000;72:2684-2689.
A stacking method is described that permits the loading of large-volume samples for capillary electrophoresis. Samples of several hundred microliters applied by electrokinetic injection are stacked at the head of a capillary electrophoresis column prior to separation. This is achieved by preceding the sample injection by pressure injection of a plug of water. The resulting nonuniform electric field causes concentration of peptide analytes at the interface between the low-conductivity water plug and the higher-conductivity electrophoresis buffer ahead of it. Low pH is used to minimize electroendosmosis so that the water plug remains near the head of the column.
Csapo Z, Gerstner A, Sasvari-Szekely M, Guttman A. Automated ultra-thin-layer SDS gel electrophoresis of proteins using noncovalent fluorescent labeling. Anal Chem 2000;72:2519-2525.
Proteins are noncovalently labeled by the addition of a fluorescent dye, Sypro Red (Molecular Probes, Eugene, OR), either during or immediately prior to sample loading. Separation is then performed in ultra-thin-layer SDS gels. These allow efficient heat removal and consequently fast separations. Detection is performed in real time by laser-induced fluorescence using an integrated scanning device. The method avoids lengthy staining procedures, is fully automated, and requires small amounts of staining dye.
Malloy MP. Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients. Anal Biochem 2000;280:1-10.
Methods for solubilizing membrane proteins for two-dimensional electrophoresis are reviewed. The use of chaotropes, new surfactants, and reducing agents are described. Gel loading strategies for use with immobilized pH gradients are summarized, and methods for selective extraction of membrane proteins are provided.
Wang E, Miller LD, Ohnmacht GA, Liu ET, Marincola FM. High-fidelity mRNA amplification for gene profiling. Nat Biotechnol 200;18:457-459.
The application of cDNA microarray is limited by the large amount of RNA required for analysis, 50 to 200 µg of total RNA or 2 to 5 µg of poly(A) RNA. This paper describes an RNA amplification method to improve the sensitivity of the technique. In the new method, RNA is used to direct the synthesis of cDNA, which in turn is amplified in an in vitro transcription reaction. One or two cycles of amplification are employed. The method used for cDNA synthesis addresses underrepresentation of 5' ends due to inefficient second-strand synthesis by employing a switch primer, and enhances the fidelity of chain synthesis by performing second-strand synthesis at the elevated temperature of 75°C. The technique yields up to 105-fold amplification of high-fidelity poly(A) RNA from nanograms of initial total RNA. This allows the possibility of using cDNA microarrays to analyze clinical specimens obtained by fine-needle aspiration or microdissection and experimental models in which embryonic tissue or small organisms are studied.
Okamoto T, Suzuki T, Yamamotot N. Microarray fabrication with covalent attachment of DNA using bubble jet technology. Nat Biotechnol 2000;18:438-441.
Microarrays are most commonly made in the academic setting by spotting probes onto glass microscope slides. Spotting is performed either with pens that contact the slide surface or with a noncontact technique. Contact printing tends to produce irregularly shaped spots and limits the attainable spot size. Reproducibility is critically important for obtaining reliable results, and it is additionally advantageous to minimize spot size to print arrays of maximal density so as to reduce the amount of starting RNA needed for analysis. A variant of the noncontact, ink-jet technology is described which uses a bubble-jet ink-jet device in which liquid is ejected from the print head by expansion of bubbles formed through heating the fluid inside. The device is used to eject oligonucleotides onto treated glass surfaces. The oligonucleotides are covalently attached to the glass via heterobifunctional cross-linkers that react with an amino group on the glass and a thiol group on the oligonucleotide. Oligonucleotide solution was ejected at 24 pl/droplet and at a resolution of 100 X 100 dpi. The spots were round and only 70 µm in diameter, which raises the possibility of manufacturing very-high-density arrays using this technique. For further commentary on this article, see also Harris TM, Massimi A, Childs G. Injecting new ideas into microarray printing. Nat Biotechnol 2000;18:384-385.