Created: 1st June 1999, last updated: 12th July 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.
Sechi S, Chait BT. Modification of cysteine residues by alkylation: a tool in peptide mapping and protein identification. Anal Chem 1998;70:5150-5158.
Cysteine residues are alkylated to increase the amount of information available for identification of proteins by peptide mass fingerprinting. Alkylation of proteins is performed prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to avoid adventitious formation of acrylamide adducts during electrophoresis. The alkylating reagents iodoacetamide, 4-vinylpyridine, and acrylamide are all successful in improving the sequence coverage for albumin. Without alkylation, peptides containing cysteine are virtually absent from the mass spectra. Cysteine alkylation is also used as a tool for spectral identification of peptides containing cysteine. A 1:1 mixture of unlabeled acrylamide and deuterium-labeled acrylamide ([2,3,3'-D3]acrylamide) is used for alkylation so as to confer characteristic isotopic distributions on cysteine-containing peptides. This additional amino acid composition information assists in protein identification.
Geyer H, Schmidt S, Wuhrer M, Geyer R. Structural analysis of glycoconjugates by on-target enzymatic digestion and MALDI-TOF-MS. Anal Chem 1999;71:476-482.
A method for structural characterization of oligosaccharides and glycoconjugates in picomole amounts is described. Samples are dried on a target plate with 6-aza-2-thiothymidine (ATT) as a matrix-assisted laser desorption/ionization (MALDI) matrix. They are then treated with exoglycosidases that have previously been dialyzed against ammonium acetate to remove salts that may interfere with ionization. The enzymes are active after dialysis and function well in the presence of the matrix. Several rounds of exoglycosidase treatments can be performed rapidly on a single spot without intermediate desalting or purification steps, thus permitting high sensitivity. MALDI-TOF is used to monitor the progress of digestion. Structural information, including monosaccharide sequence, branching pattern, and anomeric configurations of glycosidic linkages, is obtained by combining the high specificity of the enzymes with the high resolution and sensitivity of the mass analysis.
Mullikin JC, McMurray AA. Sequencing the genome, fast. Science 1999;283:1867-1868.
The performance of the new PE Biosystems Model 3700 DNA sequencer is evaluated on the basis of experience in the Sanger Center. The instrument uses an array of 96 capillaries. It is specified to be able to sequence 768 samples in a 24-hour period with less than 1 hour of human labor and uses a sheath flow fluorescence detection system with almost no moving parts. In relation to slab gel sequencers (eg, the PE Biosystems Model 377XL) the PE Biosystems Model 3700 is extremely user friendly; however, read lengths are presently shorter, although it is expected that optimization will lead to improvements in this regard. During the next 2 years, the PE Biosystems Model 3700 is likely to play a major role in the advancement of the human and other genome projects.
Iyer VR, Eisen MB, Ross DT, et al. The transcriptional program in the response of human fibroblasts to serum. Science 1999;283:83-87.
cDNA microarrays representing about 8600 different human genes (about half of them expressed sequence tags [ESTs]) were employed to document the response of fibroblasts to serum. This response is used as a model for the role of fibroblasts in wound healing. Fetal bovine serum was added to cultures of fibroblasts resting in G0 in the absence of growth factors, and changes in the expression of genes were studied over a period of 24 hours following serum addition to observe the temporal program of transcriptional changes underlying the response. Temporal profiles of gene expression were constructed using a series of 12 time points, and the "choreography" of the expression program was deduced from the resulting diverse patterns using the clustering and display method of Eisen et al. (reviewed in Bioinformatics). Although only the 517 genes that changed most strongly in transcriptional status are discussed in the printed paper, data for the complete set of 8600 genes is available at a Web site maintained by the authors' laboratory.
Green-Church KB, Limbach PA. Matrix-assisted laser desorption/ionization mass spectrometry of hydrophobic peptides. Anal Chem 1998;70:5322-5325.
Hydrophobic peptides are difficult to analyze by MALDI-MS because of their limited solubility in the aqueous solvents used in conventional matrix and sample preparation procedures. In the procedure described in this paper, hydrophobic peptides are dissolved in chloroform and combined with matrices prepared in chloroform or chloroform/methanol solutions. 2,5-Dihydroxybenzoic acid (DHB), sinapinic acid, and 3-indoleacrylic acid are suitable matrices. Because the solutions are not strongly acidic, they are suitable for peptides with acid-labile protecting groups.
DeGnore JP, Qin J. Fragmentation of phosphopeptides in an ion trap mass spectrometer. J Am Soc Mass Spect 1998;9:1175-1188.
This paper presents a systematic study of the fragmentation of phosphopeptides in an electrospray ion trap mass spectrometer. Peptides containing phosphoserine have relatively simple fragmentation patterns involving loss of H3PO4 (98 daltons), and this loss is independent of charge state. By contrast, peptides containing phosphothreonine (Thr-P) and phosphotyrosine (Tyr-P) show complicated fragmentation involving loss of H3PO4 and/or HPO3 (80 daltons), or may be detected with no loss of phosphate. Moreover, the tendency to lose phosphate from these peptides depends on charge state, with peptides in the highest charge states tending to retain phosphate while showing extensive peptide backbone fragmentation. Therefore, the best strategy for sequencing peptides containing Thr-P and Tyr-P for identification of the phosphorylation sites is to choose the species with the highest charge state, whereas for peptides containing phosphoserine (Ser-P), it is best to use the most intense peak.
Gobom J, Mirgorodskaya E, Nordhoff E, Hojrup P, Roepstorff P. Use of vapor-phase acid hydrolysis for mass spectrometric peptide mapping and protein identification. Anal Chem 1999;71:919-927.
Optimized methods of acid hydrolysis for identification of proteins by peptide mass fingerprinting are presented in this paper. Hydrolysis with 90% pentafluoropropionic acid in the gas phase at 70°C for 60 minutes in vials flushed with argon and then evacuated to 1 mbar is preferred. Addition of 1% to 5% dithiothreitol (DTT) suppresses oxidation of Met and Tyr. Under such conditions, cleavages at Asp, Ser, Thr, and Gly, and N- and C-terminal sequence ladders predominate. Numbers of cleavages are relatively small, and deamidation of Asn and Gln is not observed. The method can be used on proteins in destained, dried gel pieces after SDS-PAGE.
Johnson KL, Veenstra TD, Londowski JM, Tomlinson AJ, Kumar R, Naylor S. On-line sample clean-up and chromatography coupled with electrospray ionization mass spectrometry to characterize the primary sequence and disulfide bond content of recombinant calcium binding proteins. Biomed Chromatogr 1999;13:37-45.
The presence of salts, buffers, denaturants, and detergents often interfere with electrospray ionization (ESI). In this paper, a system is described for on-line removal of such substances which consists of a pressurized bomb used to apply sample through a fused silica capillary transport tube onto a microcolumn of solid, reverse-phase packing material. The protein is washed, eluted from the microcolumn, and passed through another capillary transport tube to the ESI interface. This approach permits accurate mass determination of proteins from solutions with a wide variety of interfering constituents.
Lozano JM, Espejo F, Diaz D, et al. Reduced amide pseudopeptide analogues of a malaria peptide possess secondary structural elements responsible for induction of functional antibodies which react with native proteins expressed in Plasmodium falciparum erythrocyte stages. J Pept Res 1998;52:457-469.
Pseudopeptides with the sequence KEKMV were synthesized. This sequence is derived from the merozoite surface protein (MSP-1) of the malaria parasite and is involved in the attachment of the malarial parasite to human erythrocytes. One peptide bond at a time was replaced by a psi[CH2NH] bond by condensing a protected amino acid aldehyde with resin-bound peptide in the presence of the reducing agent NaBH3CN. Monoclonal antibodies were made against the resulting peptide analogs and shown to cross-react with native MSP-1, modify the kinetics of the parasite's development inside the red blood cell, and reduce the parasite's ability to invade new red blood cells. Pseudopeptides therefore represent candidates for the development of malarial vaccines.
Aguilar RM, Bustamante JJ, Hernandez PG, Martinez AO, Haro LS. Precipitation of dilute chromatographic samples (ng/ml) containing interfering substances for SDS-PAGE. Anal Biochem 1999;276:344-350.
SDS-PAGE analysis of very dilute proteins and proteins accompanied by various interfering substances is problematic. In this paper, a method for precipitating proteins with the dye pyrogallol red-molybdate is shown to be effective for concentrating proteins from solutions containing as little as 7 ng/ml of protein. This method works in the presence of a wide variety of interfering substances, including organic solvents, chaotopes, sucrose, ethylenediaminetetraacetic acid (EDTA), high salt (1.0 M NaCl), and nonionic and zwitterionic detergents, but is not efficient in the presence of ammonium sulphate or ionic detergents such as SDS. It successfully precipitated some proteins that were difficult to precipitate with trichloroacetic acid or phenol-ether.
Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 198;95:14863-14868.
A first step in extracting gene expression information from DNA microarrays is to identify extremes of differential expression in two individual samples or in a time series after a given treatment. This paper extends the information that can be extracted by proposing a method for organizing the entire set of observations in a holistic fashion. Tables of data are ordered by clustering genes with similar patterns of expression (eg, similar shapes of the curves describing the rise and fall of mRNA abundance in a time series). The result is a hierarchical clustering similar to phylogenetic analysis of sequences. Clustering is combined with a graphic representation in which each data point is represented by a color that quantitatively and qualitatively reflects the original observations. The method is applied to time courses of mitotic cell division, sporulation, the diauxic shift, and shock responses in yeast, and to the response of mammalian, serum-deprived cells to added serum (see review of Iyer et al. in Genes--Cloning, Sequencing, and Expression). The clustering method is found to group together genes that have similar functions. This allows the possibility of gaining insight into the functions of poorly characterized or novel genes by studying their expression patterns.
Angeletti RH, Bonewald LF, De Jongh K, Niece R, Rush J, Stults J. Research technologies: fulfilling the promise. FASEB J 1999;13:595-601.
Issues concerning the establishment and maintenance of shared technology resource (ie, "core") laboratories are considered in this paper. These laboratories provide the technologic infrastructure on which contemporary experimental biology depends, but local resource laboratories are costly, their intellectual base requires ongoing support, and they require long-term institutional commitment. To be successful, therefore, they require an active partnership among scientists, administration, and funding agencies. Fee structure, availability of special funds for project start-up, continuing training and intellectual involvement of laboratory staff, and plans for long-term investment and commitment are all critical factors in the success of a core laboratory. Examples of harmful as well as helpful policies with respect to these factors are considered.