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 Associates to forward information on articles they feel are important and useful to any member of the Editorial Board.
Amino Acid and Sequence Analysis
Bank, R. A., Jansen, E. J., Beekman, B. and te Koppele, J. M. (1996) Analytical Biochemistry 240, 167-176. Amino Acid Analysis by Reverse-Phase High-Performance Liquid Chromatography: Improved Derivatization and Detection Conditions with 9-Fluorenylmethyl Chloroformate.
Tweaking the standard Fmoc amino acid analysis procedure resulted in several substantial improvements. Derivatization for 40 minutes in a pH 11.4 borate buffer reduced interference from reagent hydrolysis and provided derivatives that were stable for at least 48 hours. His and Tyreach converted to two derivatives in the standard procedureproduced single derivatives. Using an emission wavelength of 630 nm instead of 313 or 340 nm lowered baseline noise without sacrificing sensitivity.
Iwamatsu, A. and Yoshida-Kubomura, N. (1996) Journal of Biochemistry 120, 29-34. Systematic Peptide Fragmentation of Polyvinylidene Difluoride (PVDF)-Immobilized Proteins Prior to Microsequencing.
Peptides are recovered at increased yields when PVDF-bound proteins stained with Ponceau S are sequentially digested with a battery of proteases, presumably because fewer large, hydrophobic peptides are left on the membranes. Membrane-bound proteins were digested with Achromobacter protease I, endoproteinase Asp-N, and trypsin (in this order), and the supernatant after each digestion was fractionated by HPLC. Proteins with molecular weights up to 400,000 were successfully fragmented, and yields from 50 pmol of sample approached 80%.
Capillary Electrophoresis
Shihabi, Z. K. (1996) Journal of Chromatography A 744, 231-240. Peptide Stacking by acetonitrile-salt mixtures for capillary zone electrophoresis.
Before capillary electrophoresis, peptide samples were concentrated as much as 20-fold simply by dissolving in two volumes of acetonitrile and one volume of 1% NaCl. Proteins precipitated in this mixture and were separated from peptides, useful for analysis of proteolytic peptides. The mixture can be loaded directly into capillaries and can occupy as much as one-third the capillary volume, without loss of sample or resolution. During electrophoresis peptides are stacked by an unknown mechanism, apparently different from stacking in aqueous, low ionic strength buffers.
DNA Synthesis
Pon, R. T., Buck, G. A., Hager, K. M., Naeve, C. W., Niece, R. L., Robertson, M. and Smith, A. J. (1996) BioTechniques 21, 680-685. Multi-Facility Survey of Oligonucleotide Synthesis and an Examination of the Performance of Unpurified Primers in Automated DNA Sequencing.
Results of the most recently completed study conducted by the ABRF Nucleic Acids Research Committee, based on analysis of 208 samples submitted to the Committee by 71 study participants. Evaluation by capillary electrophoresis showed that for 85% of these samples coupling efficiencies during synthesis exceeded 98%. When unpurified 25-base samples were used as primers for automated DNA sequencing, there was no correlation between oligonucleotide purity and sequence accuracy, as long as the primers were at least 70% pure.
DNA Sequencing and Analysis
Adams, P. S., Dolejsi, M. K., Hardin, S., Mische, S., Nanthakumar, B., Riethman, H., Rush, J. and Morrison, P. (1996) BioTechniques 21, 678. DNA Sequencing of a Moderately Difficult Template: Evaluation of Results from a Thermus thermophilus Unknown Test Sample.
A summary of the first study conducted by the ABRF DNA Sequence Research Committee, based on 73 datasets submitted by 50 study participants. The study results are described in detail at a homepage established by this Committee, at the URL address: http:/ /mbcf.dfci.harvard.edu/abrfdnaseq.
Hall, J. M., LeDuc, C. A., Watson, A. R. and Roter, A. H. (1996) Genome Research 781-790. An Approach to High-throughput Genotyping.
A short, thorough, and practical review on automated genotyping with sections on current technologies, data management, common genotyping problems, future improvements, and one case study. The review is based on the experiences of a high-throughput facility capable of generating more than two million genotypes per year.
Parker, L. T., Zakeri, H., Deng, Q., Spurgeon, S., Kwok, P.-Y. and Nickerson, D. A. (1996) BioTechniques 21, 694-699. AmpliTaq DNA Polymerase, FS Dye-Terminator Sequencing: Analysis of Peak Height Patterns.
For practical reasons many laboratories prefer automated cycle -sequencing with unlabeled primers and dye-labeled ddNTP terminators, even though dye-terminators produce less uniform peak heights than dye-labeled primers. This study systematically examined dye-terminator data to learn how sequence context influences peak heights. Remarkably, much of the peak height variation is due to short-range effects: in 31 of the 64 possible triplet sequences, the peak height of a base could be predicted by knowing just one or two upstream bases. With or without incorporation in sequence analysis algorithms, awareness of these patterns improves sequencing accuracy.
Bentzley, C. M., Johnston, M. V., Larsen, B. S. and Gutteridge, S. (1996) Analytical Chemistry 68, 2141-2146. Oligonucleotide Sequence and Composition Determined by Matrix-Assisted Laser Desorption /Ionization.
In this report, MALDI-MS was used to sequence 100 pmol quantities of oligonucleotides up to 24 bases long. Samples were digested with calf spleen phosphodiesterase for sequential cleavage at the 5' end, and 10 pmol amounts of digested oligonucleotides were mixed with 3 -hydroxypicolinic acid matrix containing ammonium citrate, to minimize cation adducts. Visualization of samples during analysis suggested the strongest signals were produced by irradiating the edges of sample-matrix crystals.
Smirnov, I. P., Roskey, M. T., Juhasz, P., Takach, E. J., Martin, S. A. and Haff, L. A. (1996) Analytical Biochemistry 238, 19-25. Sequencing Oligonculeotides by Exonuclease digestion and Delayed Extraction Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry.
A similar report on sequencing based on use of 5'- and 3'-exonucleases followed by MALDI-MS. Performing 5'-exonuclease digestions directly in 3'-hydroxypicolinic acid:ammonium citrate matrix allowed analysis of 20 pmol quantities of a 33-base oligonucleotide. The authors show delayed extraction greatly improves spectrum quality increasing resolution to about 3,000and claim the method "is now routinely applied to oligonucleotides as long as 50 bases".
Roskey, M. T., Juhasz, P., Smirnov, I. P., Takach, E. J., Martin, S. A. and Haff, L. A. (1996) Proceedings of the National Academy of Sciences (USA) 93, 4724-4729. DNA sequencing by delayed extraction -matrix-assisted laser desorption/ionization time of flight mass spectrometry.
The same group uses delayed extraction MALDI to analyze enzymatic dideoxy cycle sequencing products. A 13-base primer was annealed to 40- or 50-base templates, and cycle sequencing conditions were optimized to give as much as 100 fmol of each extension product. Reaction mixtures were mixed with 3-hydroxypicolinic acid: ammonium citrate matrix and applied to Nafion-coated targets. A full sequencing dataset is shown for the 50-base template only, and it can be interpreted but non-specific base terminations and weak signals interfere significantly.
Ni, J., Pomerantz, S. C., Rozenski, J., Zhang, Y. and McCloskey, J. A. (1996) Ananlytical Chemistry 68, 1989-1999. Interpretation of Oligonucleotide Mass Spectra for Determination of Sequence Using Electrospray Ionization and tandem Mass Spectrometry.
In this report, oligonucleotides up to 10 bases long were electrospray ionized and sequenced by induced fragmentation in a quadrupole collision cell. No attempt was made to determine the lower limit for analysis, but typically oligonucleotide concentrations were 20 pmol /ml and volumes were less than 10 ml.
Li, Y., Tang, K., Little, D. P., Köster, H., Hunter, R. L. and McIver, R. T., Jr. (1996) Analytical Chemistry 68, 2090-2096. High-Resolution MALDI Fourier Transform Mass Spectrometry of Oligonucleotides.
In this report, oligonucleotides were ionized by MALDI at an external source and then shunted to a superconducting magnet Fourier transform analyzer, where mass is measured from coherent resonance frequencies. Moving ions from the source to the analyzer with a low -energy ion guide and cooling the ions with a pulse of argon overcame previous limitations due to poor sensitivity and inadvertent fragmentation. The authors used this technology to analyze several oligonucleotides 6-12 bases long; with 12 pmol of a 25-base oligonucleotide, they obtained a mass resolution of 136,000.
Mass Spectrometry
Carr, S. A., Huddleston, M. J. and Annan, R. S. (1996) Analytical Biochemistry 239, 180-192. Selective Detection and Sequencing of Phosphopeptides at the Femtomole Level by Mass Spectrometry.
A nanospray mass analysis method is described for identifying phosphorylated residues in unfractionated proteolytic digests. Femtomole quantities of peptides were analyzed according to a systematic, four-step procedure. Little material was consumed, allowing further downstream processing, and phosphopeptide sequence information was obtained with 50 to 100 times less sample than needed for Edman sequence analysis.
Annan, R. S. and Carr, S. A. (1996) Analytical Chemistry 68, 3413 -3421. Phosphopeptide Analysis by Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry.
Similar to the previous citation, but here MALDI-TOF was used to identify and sequence subpicomole amounts of HPLC-purified phosphopeptides. With peptides containing a single phosphate group, reflectron analysis distinguished pS/pT from pY: peptides containing pY produced [MH-HPO3]+ species (-79 Da), and peptides containing pS or pT gave [MH-H3PO4]+ species (-97 Da). The authors claim this analysis can be performed routinely with about 300 fmol of phosphopeptide.
Zhang, X., Dillen, L., Vanhoutte, K., Van Dongen, W., Esmans, E. and Claeys, M. (1996) Analytical Chemistry 68, 3422-3430. Characterization of Unstable Intermediates and Oxidized Products Formed during Cyanogen Bromide Cleavage of Peptides and Proteins by Electrospray Mass Spectrometry.
In this report CNBr digestion products were investigated by mass analysis. Endorphins and myoglobin were cleaved with CNBr in 0.1% TFA/acetonitrile (6/4, v/v), in 70% formic acid, or in 70% TFA. All three cleavage conditions produced the expected fragments and methionine-oxidized peptides. Interestingly, under certain analytical conditions a cyclic hydrated homoserine iminolactone intermediate was observed, which is thought to be responsible for poor cleavage of Met-Thr bonds.
Protein Characterization and Analysis
Cornish, W. V., Hahn, K. M. and Schultz, P. G. (1996) Journal of the American Chemical Society 118, 8150-8151. Site-Specific Protein Modification Using a Ketone Handle.
A reactive ketone group was bioengineered at specific sites of T4 lysozyme by using E. coli transcription/translation extracts containing amber suppressor tRNAs charged with a phenolic ketone amino acid analogue. The ketone group is highly reactive toward alkoxyamines and hydrazides and so provides a site for directed labeling with reagents such as fluorescein hydrazide. The authors foresee novel applications including the engineering of protein biosensors that can report on the environment of living cells.
Wen, J., Arakawa, T. and Philo, J. S. (1996) Analytical Biochemistry 240, 156-166. Size-Exclusion Chromatography with On-Line Light -Scattering, Absorbance, and Refractive Index Detectors for Studying Proteins and Their Interactions.
A review of applications for SEC-HPLC configured with serial ultraviolet absorbance, refractive index, and light-scattering detectors, with emphasis on proteins/glycoproteins and non-covalent protein complexes. Under appropriate conditions, on-line light scattering provides an absolute measurement of molecular weight, independent of particle size and elution time and without the need for calibration with molecular weight standards. This technology allows analysis of non-covalent complexes under physiological conditions. The examples described in this review include antigen-antibody complexes, ligand -receptor complexes (tumor necrosis factor, neurotrophic factor, and neurotrophin-3), and heparin-basic fibroblast growth factor complexes.
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