This column highlights several recently published articles that are likely to be of interest to the readership of this newsletter. Articles are selected for listing and summarized by some members of the Editorial Board. Article summaries reflect their opinions and not necessarily those of the Association. We encourage ABRF members to forward information on articles they feel are important and useful to any member of the Editorial Board.
Amino Acid and Sequence Analysis
Nokihara, K., Morita, N., Yamamoto, R. and Kondo, J. (1995) Journal of Biochemistry 118, 895-899. Instrumentation and Applications of an Automated Carboxyl-Terminal Fragment Peptide Fractionator for C -Terminal Sequence Analysis of Proteins.
Proteins are digested with Lys-C and the resulting peptides covalently immobilized through their a- and e-amino groups to an Edman reagent analogue, para-phenylene diisothiocyanate, on beads. The carboxyl -terminal peptide, which lacks lysine, is then the only peptide that is not "double-anchored" to the beads. This peptide, minus its amino-terminal residue, is then selectively released through an Edman-like cyclization and cleavage step by treating the beads with trifluoroacetic acid.
Wilm, M., Shevchenko, A., Houthhaeve, T., Breit, S., Schweigerer, L., Fotsis, T. and Mann, M. (1996) Nature 379, 466-469. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.
Stellar report possibly signaling the death knell for many popular applications of Edman sequencing. This paper shows convincingly that about 5 ng of protein from stained gels can provide sequence information by MS/MS analysis. However, the method requires unique, sophisticated instrumentation that is not yetbut is likely to becomecommercially available.
Shevchenko, A., Wilm, M., Vorm, O. and Mann, M. (1996) Analytical Chemistry 68, 850-858. Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels.
Similar to the reference above, but giving a more detailed account of the instrumentation and experimental design. Instead of separating peptides by HPLC with on-line MS for analysis, unseparated digests are infused as mixtures into the mass spectrometer using a fine needle. Each sample can be analyzed for up to one hour, providing time for optimization of individual peptide spectra. Several examples are presented, demonstrating persuasively that low-level sequencing is now a reality.
Simpson, J.T., Torok, D.S., Girard, J.E. and Markey, S.P. (1996) Analytical Biochemistry 233, 58-66. Analysis of Amino Acids in Biological Fluids by Pentafluorobenzyl Chloroformate Derivatization and Detection by Electron Capture Negative Ionization Mass Spectrometry.
Pentafluorobenzyl chloroformate amino acids are readily detected at femtomole levels by gas chromatography/electron-capture negative ionization mass spectrometry and selective ion-monitoring of the [M -181] negative ion. After a simple, one-step derivatization procedure, a wide range of samples (free amino acids, protein acid hydrolyzates, plasma, and whole blood) can be successfully analyzed.
Ou, K., Wilkins, M.R., Yan, J.X., Gooley, A.A., Fung, Y., Sheumak, D. and Williams, K.L. (1996) Journal of Chromatography A 723, 219-225. Improved high-performance liquid chromatography of amino acids derivatised with 9-fluorenylmethyl chloroformate.
Continuing research by this group to improve Fmoc amino acid analysis. Multi-tasking computer control is used to optimize peak resolution,
increase sample throughput, and improve chromatographic reproducibility. Furthermore, this allows column regeneration and reduces the cost of analysis to about $1 per run. Examples include protein analysis from two-dimensional gel spots blotted to PVDF.
Carbohydrate Structure and Analysis
Rassi, Z.E. (Editor) (1996) Electrophoresis 17, 273-440.
Special issue devoted to Capillary Electrophoresis of Carbohydrate Species.
Geise, R. and Honda, S. (Editors) (1996) Journal of Chromatography A 720, 1-447.
Special Issue devoted to Chromatographic and Electrophoretic Analyses of Carbohydrates.
Yang, Y. and Orlando, R. (1996) Analytical Chemistry 68, 570-572. Simplifying the Exoglycosidase Digestion/MALDI-MS Procedures for Sequencing N-Linked Carbohydrate Side Chains.
Many exoglycosidase digestions use buffers containing sodium counterions, but sodium can interfere during MS analysis of the unfractionated mixture through adduct formation. This report shows that ammonium acetate can replace the usual sodium-counterion buffers without loss of enzyme activity or specificity. When digests in ammonium acetate are analyzed by MALDI using sinapinic acid as the matrix, protonated ions are observed without interference from sodium adducts.
DNA Sequencing
Saccharomyces Genome Database, Stanford University School of Medicine. URL address: http://genome-www.stanford.edu/Saccharomyces/
On April 24, 1996 the first complete genomic sequence of a eukaryotic organism was released, that of Saccharomyces cerevisiae strain S228C, the results of an international collaboration of more than 100 laboratories. The genome of the organism contains 13,105,020 base pairs. Go to this site on the WWW to read the details and press releases.
Metzker, M.L., Lu, J. and Gibbs, R.A. (1996) Science 271, 1420-1422. Electrophoretically Uniform Fluorescent Dyes for Automated DNA Sequencing.
The fluorescent dyes currently used for automated DNA sequencing are based on the old standbys fluoroscein and rhodamine but are less than ideal: their fluorescence emission spectra overlap (allowing crosstalk), and they alter electrophoretic mobilities in dye-specific manners. Because of this, raw data must be "corrected" with software before it can be used. This report shows these shortcomings do not apply to primers labeled with BODIPY dyes. Unprocessed data can be read directly, impacting general DNA sequencing and heterozygote detection. And higher fluorescence intensities should result in lower reagent costs than conventional dyes.
Ju, J., Glazer, A.N. and Mathies, R.A. (1996) Nature Medicine 2, 246-249. Energy transfer primers: A new fluorescence labeling paradigm for DNA sequencing and analysis.
Another strategy for improving on conventional fluorescent dyes, this one based on fluorescence energy transfer. Here primers are made with a fluorescein donor-dye at the 5' end and one of the four conventional dyes 10 bases downstream, acting as acceptors: when the donor absorbs and emits light, some of the emitted light is absorbed by the acceptor and re -emitted at a different wavelength. These double-labeled primers emit 2 -14 times more intensely than their single-dye counterparts. An energy transfer cassettea phosphodiester backbone with the donor and acceptor dyes but without basescan be attached to the 5' end of custom sequencing primers.
Mass Spectrometry
Zhang, H., Andrén, P.E. and Caprioli, R.M. (1995) Journal of Mass Spectrometry 30, 1768-1771. Micro-Preparation Procedure for High -Sensitivity Matrix-Assisted Laser Desorption Ionization Mass Spectrometry.
A fused-silica C18-packed capillary and multiport valve were used to concentrate, desalt, and add matrix to trace quantities of peptides before MALDI mass analysis. This method for sample preparation allowed mid-attomol detection of peptides and was used to analyze hormone metabolites levels during in vivo microdialysis.
Cohen, S.L. and Chait, B.T. (1996) Analytical Chemistry 68, 31-37. Influence of Matrix Solution Conditions on the MALDI-MS Analysis of Peptides and Proteins.
MALDI-MS analysis of mixtures, when using the popular matrix a-cyano-4-hydroxycinnamic acid, sometimes does not show all species present in the mixture, presumably because of selective ionization. This detailed study shows selective ionization is influenced both by the composition of the sample-matrix solution and by the growth kinetics of sample-matrix co-crystals. Samples analyzed under one set of conditions generally cannot be considered adequately analyzed. Guidelines are given for various sample types.
Gharahdaghi, F., Kirchner, M., Fernandez, J. and Mische, S.M. (1996) Analytical Biochemistry 233, 94-99. Peptide-Mass Profiles of Polyvinylidene Difluoride-Bound Proteins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry in the Presence of Nonionic Detergents.
Continuing work from this group on the effect of nonionic detergents on various protein analytical methods. In this report, use of a modified matrix solution (50% acetonitrile/0.05% TFA saturated with a-cyano-4-hydroxycinnamic acid) during MALDI/MS analysis of PVDF-bound proteins proteolyzed in the presence of detergentsincluding hydrogenated Triton X-100, octylglucoside, and Tween 20was found to alleviate the signal suppression associated with unmodified matrix solutions. High quality spectra of unfractionated mixtures at low pmol amounts were obtained only when using the modified matrix solution.
PeptidesChemistry and Purification
Smart, S.S., Mason, T.J., Bennell, P.S., Maeij, N.J. and Geysen, H.M. (1996) International Journal of Peptide and Protein Research 47, 47-55. High-throughput purity estimation and characterisation of synthetic peptides by electrospray mass spectrometry.
High-throughput analysis of target peptides and potential by-products after Multipin solid-phase synthesis. Electrospray mass spectra are analyzed using an algorithm that calculates the expected mass of the target and the mass of by-products that could occur from known side -reactions. Thus, it is possible in one run to provide information on the quality of these synthetic peptides frequently used in peptidomimetics and to design synthesis protocols to minimize reaction by-products.
Protein Characterization and Analysis
Funderburgh, J.L. and Prakash, S. (1996) Biotechniques 20, 376-378. SDS-Polyacrylamide Gel Electrophoretic Analysis in the Presence of Guanidinium Hydrochloride.
It is often necessary to use the powerful chaotropic agent guanidinium hydrochloride to solubilize and extract proteins and peptides. Unfortunately, this reagent is not compatible with SDS-PAGE. A simple procedure that both removes the chaotrope and concentrates samples is presented. The protein is adsorbed to nitrocellulose, washed, stained
with Ponseau S, and subsequently eluted at 70°C with a minimal volume of SDS-PAGE loading buffer. Many samples can be processed in a 96 -well format if desired, and the procedure may be applicable to other problematic protein additives.
Liang, X., Bai, J., Liu, Y.-H. and Lubman, D.M. (1996) Analytical Chemistry 68, 1012-1018. Characterization of SDS-PAGE Separated Proteins by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.
Proteins or CNBr fragments separated by SDS-PAGE are electroblotted to nitrocellulose. The membrane is stained with Ponseau S and is then intentionally dissolved with a matrix/acetone solution before MALDI -MS analysis. This one-step procedure allows high recoveries, and some in situ digestions are also possible, facilitating peptide mapping experiments.
Sulfhydryl Analysis
Wu, J., Gage, D.A. and Watson, J.T. (1996) Analytical Biochemistry 235, 161-174. A Strategy to Locate Cysteine Residues in Proteins by Specific Chemical Cleavage Followed by Matrix-Assisted Laser Desorption /Ionization Time-of-Flight Mass Spectrometry.
A MALDI-MS procedure for proteins with unknown sulfhydryl oxidation state but known sequence is described. The method counts cysteine and, when coupled with sample reduction, infers the number of cystine residues (but generally cannot assign disulfide connectivity). Free sulfhydryl groups are cyanylated with 2-nitro-5-thiocyanobenzoic acid and subsequently cleaved by exposure to mild alkaline buffers. The fragmented sample is then reduced and mass analyzed to give total cysteine content. Concurrently, a second sample is reduced before cyanylation, thereby yielding total cysteine plus cystine content. Major advantages include low sample consumption and rapid assignment of sulfhydryl oxidation state.
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