Article Watch

Amino Acid and Sequence Analysis

• Kawakami, Y. and Ohmori, S. (1994) Analytical Biochemistry 220, 66-72. "Microidentification of N-Terminal-Blocked Amino Acid Residues of Proteins and Peptides."
  Improvements on previous work using protease digestion of the blocked sample followed by precolumn carboxyl esterification with 9-anthryldiazomethane and HPLC analysis. Sensitivity is reported in the low pmol range for several N-terminal blocked samples.
• Strydom, D. J. and Cohen, S. A. (1994) Analytical Biochemistry 222, 19-28. "Comparison of Amino Acid Analyses by Phenylisothiocyanate and 6-Aminoquinolyl-N-HydroxySuccinmidyl Carbamate Precolumn Derivatization."
  Thorough review comparing two high sensitivity precolumn amino acid derivatization procedures. Advantages and disadvantages of each are explored.
• Reynolds, E. C., Riley, P. F. and Adamson, N. J. (1994) Analytical Biochemistry 217, 277-284. "A Selective Precipitation Purification Procedure for Multiple Phosphoseryl-Containing Peptides and Methods for Their Identification."
  Calcium and ethanol solutions at aqueous pH values of 3.5, 4.6, and 8.0 differentially precipitated contiguous from non-contiguous phosphopeptides derived from proteolytic digests. A minimal phosphoseryl motif is described for successful application of this procedure.
• van der Geer, P. and Hunter, T. (1994) Electrophoresis 15, 544-554. "Phosphopeptide mapping and phosphoamino acid analysis by electrophoresis and chromatography on thin-layer cellulose plates."
  A "how-to" guide to 2D phosphopeptide mapping analysis by a leading laboratory. Thorough presentation on the advantages and limitations of this procedure.

Capillary Electrophoresis

• Guttman, A. and Nolan, J. (1994) Analytical Biochemistry 221, 285-289. "Comparison of the Separation of Proteins by Sodium Dodecyl Sulfate-Slab Gel Electrophoresis and Capillary Sodium Dodecyl Sulfate-Gel Electrophoresis."
  Denaturing electrophoretic analysis of 65 proteins showed comparable mobility-log MW relationships in gels (R2 = 0.973) and in capillaries (R2 = 0.995) thereby establishing capillary-SDS as an equally valid method for the estimation of protein molecular weights.
• Rudnick, S. E., Hilser, V. J. Jr. and Worosila, G. D. (1994) Journal of Chromatography A 672, 219-229. "Comparison of the utility of capillary zone electrophoresis and high-performance liquid chromatography in peptide mapping and separation."
  Head-to-head study evaluating RP-HPLC and CZE for proteolyzed recombinant proteins. Useful information that could be incorporated into GLP or GMP procedures.
• Benedek, K. and Guttman, A. (1994) Journal of Chromatography A 680, 375-381. "Ultra-fast high-performance capillary sodium dodecyl sulfate gel electrophoresis of proteins."
  A three-minute separation at high field strength is developed for proteins ranging in MW from 14 to 95 kDa using a 50 mm diameter uncoated fused-silica capillary in the presence of polyethylene oxide. Several parameters including SDS concentration and sample treatment are investigated.

DNA Sequencing

• Kotler, L., Sobolev, I. and Ulanovsky, L. (1994) BioTechniques 17, 554-559. DNA Sequencing: Modular Primers for Automated Walking.
  A DNA sequencing method based on use of modular primers assembled from 5-, 6-, and 7-mers selected from a presynthesized library of 1000 oligonucleotides. This methods uses the T7 DNA polymerase/dye-terminator automated sequencing chemistry, reduces delays during walking, and significantly lowers the cost of primer-walking sequencing strategies.
• Swerdlow, H., Dew-Jager, K. and Gesteland, R. F. (1994) BioTechniques 16, 684-93. Reloading and stability of polyacrylamide slab gels for automated DNA sequencing.
  The stability of various slab gel formulations and their ability to survive multiple loadings with sequencing samples are compared. Gels containing formamide are shown to be superior to their urea counterparts and are reusable for at least four consecutive runs.

Other Applications for Automated DNA Sequencers

• Schwengel, D. A., Jedlicka, A. E., Nanthakumar, E. J., Weber, J. L. and Levitt, R. C. (1994) Genomics 22, 46-54. Comparison of fluorescence-based semi-automated genotyping of multiple microsatellite loci with autoradiographic techniques.
  A fluorescence-based genotyping protocol is compared to standard radiolabeling methods. With fluorescence technology, up to 24 highly polymorphic microsatellite markers can be analyzed simultaneously on a single gel.

DNA Synthesis

• Hardy, P. M., Holland, D., Scott, S., Garman, A. J., Newton, C. R. and McLean, M. J. (1994) Nucleic Acids Research 22, 2998-3004. Reagents for the preparation of two oligonucleotides per synthesis (TOPS).
  Novel phosphoramidite reagents are described for synthesis of two oligonucleotides on one column. These may be useful in applications where oligonucleotides are used in pairs, such as PCR and chemical synthesis of genes.

Mass Spectrometry

• Bartlett-Jones, M., Jeffery, W. A., Hansen, H. F. and Pappin, D. J. C. (1994) Rapid Communications in Mass Spectrometry 8, 737-742. "Peptide Ladder Sequencing by Mass Spectrometry Using a Novel, Volatile Degradation Reagent."
  Refinement of the Chait et al. ladder sequencing procedure via design and synthesis of a volatile Edman reagent. Data are presented for several peptides at the low pmol level.
• Weinmann, W., Parker, C.E., Deterding, L.J., Papac, D.I., Hoyes, J., Przybylski, M. and Tomer, K.B. (1994) Journal of Chromatography A 680, 353-361. "Capillary electrophoresis-matrix-assisted laser-desorption ionization mass spectrometry of proteins."
  An "off-line" CE isolation procedure prior to MALDI using a sheath flow interface is reported. Direct analysis of proteins near 70 kDa at about 1 pmol appears possible.
• Thiede, B., Wittmann-Liebold, B., Bienert, M. and Krause, E. (1994) FEBS Letters 357, 65-69. "MALDI-MS for C-terminal sequence determination of peptides and proteins degraded by carboxypeptidase Y and P."
  Combined use of carboxypeptidase Y and P allows a "C-terminal peptide ladder" procedure. Up to 11 residues were correctly assigned from the C-terminus of known samples at pmol sensitivity.
• Vorm, O. and Roepstorff, P. (1994) Biological Mass Spectrometry 23, 734-740. "Peptide Sequence Information Derived by Partial Acid Hydrolysis and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry."
  Limited HCl hydrolysis on pmol amounts of material can provide partial sequence information following MALDI-MS analysis. Fourteen peptides ranging in size from 8 to 42 residues were studied and partial sequence was obtained for all but one.
• Kouach, M., Belalche, D., Jaquinod, M., Couppez, M., Kmiecik, D., Ricart, G., Van Dorsselael, A., Sauti¸re, P. and Briand, G. (1994) Biological Mass Spectrometry 23, 283-294. "Application of Electrospray and Fast Atom Bombardment Mass Spectrometry to the Identification of Post-Translational and other Chemical Modifications of Proteins and Peptides."
  General, up-to-date description on the use of ES-MS and FAB-MS for protein and peptide analysis with particular emphasis on identification of modified amino acids.

Nucleic Acid Services

• Pon, R. T., Buck, G. A., Niece, R. L., Robertson, M., Smith, A. J. and Spicer, E. (1994) BioTechniques 17, 526-534. "A Survey of Nucleic Acid Services in Core Laboratories."
  Results of ABRF's survey of 85 facilities offering DNA synthesis and 35 facilities offering DNA sequencing services. Data on volume, costs, number of users, instrumentation, and methodologies are compiled.

Peptides-Chemistry and Purification

• Rietman, B. H., Smulders, R. H. P. H., Eggen, I. F., Van Vliet, A., Van De Werken, G. and Tesser, G. I. (1994) International Journal of Peptide and Protein Research 44, 199-206. "Protected peptide disulfides by oxidative detachment from a support."
  Formation of cyclic disulfide or protected cysteine peptide products depended on the solvent used during iodolytic cleavage of Fmoc synthesized peptides. Some degree of selecting disulfide partners may be possible.
• Hansen, P. and Lindeberg, G. (1994) Journal of Chromatography A 662, 235-241. "Purification of tryptophan containing synthetic peptides by selective binding of the a-amino group to immobilised metal ions."
  Metal affinity chromatography is used to bind tryptophan containing synthetic peptides with a free N-terminus in the presence of Cu2+ or Ni2+ ions.
• Turck, C. W. and Edenson, S. P. (1994) Peptide Research 7, 140-145. "Mapping of Tyrosine Kinase Autophosphorylation Sites with Synthetic Peptide Substrates."
  Streamlined peptide chemical approach to delineate possible tyrosine phosphorylation sites in a protein kinase. Once identified, these tyrosine phosphopeptides facilitate in vitro and in vivo site determination.

Protein Characterization and Analysis

• Jones, M. D., Merewether, L. A., Clogston, C. L. and Lu, H. S. (1994) Analytical Biochemistry 216, 135-146. "Peptide Map Analysis of Recombinant Human Granulocyte Colony Stimulating Factor: Elimination of Methionine Modification and Nonspecific Cleavages."
  A "how-to" study on the optimization of proteolytic mapping of a recombinant protein in a GLP/GMP environment.
• Fernandez, J., Andrews, L. and Mische, S. M. (1994) Analytical Biochemistry 218, 112-117. "An Improved Procedure for Enzymatic Digestion of Polyvinylidene Difluoride-Bound Proteins for Internal Sequence Analysis."
  Further work by these authors showing that a one-step treatment with RTX-100 effectively substitutes for PVP-40 during the proteolytic digestion of PVDF-bound proteins. Pmol sensitivity is demonstrated.
• Elicone, C., Lui, M., Geromanos, S., Erdjument-Bromage, H. and Tempst, P. (1994) Journal of Chromatography A 676, 121-137. "Microbore reversed-phase high-performance liquid chromatographic purification of peptides for combined chemical sequencing-laser-desorption mass spectrometric analysis."
  Thorough practical account by a leading laboratory in this field on the optimization of HPLC components, sample manipulation, and downstream analysis of synthetic or proteolytically derived peptides at pmol levels.
• Affolter, M., Watts, J. D., Krebs, D. L. and Aebersold, R. (1994) Analytical Biochemistry 223, 74-81. "Evaluation of Two-Dimensional Phosphopeptide Maps by Electrospray Ionization Mass Spectrometry of Recovered Peptides."
  Description on the use of and interpretation of results from 2D phosphopeptide maps to isolate samples for ESI-MS at near fmol levels. A chromatographic procedure to enrich samples is also given.

Sulfhydryl Analysis

• Jue, R.A. and Hale, J.E. (1994) Analytical Biochemistry 221, 374-378. "On-Line Procedures for Alkylation of Cysteine Residues with 3-Bromopropylamine prior to Protein Sequence Analysis."
  Continued assessment by these authors on cysteine modification via 3-bromopropylamine. This article describes the in situ reduction and alkylation of samples prior to N-terminal sequence analysis on an Applied Biosystems 477A instrument.
• Shimada, K. and Mitamura, K. (1994) Journal of Chromatography B 659, 227-241. "Derivatization of thiol-containing compounds."
  Thorough review describing useful clinical/biochemical thiol derivatization chemistries. Several different detection modes are discussed.

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