ARTICLE WATCH

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.

DNA Sequencing and Analysis

 
• Ball, R.W. and Packman, L.C. Analytical Biochemistry 246, 185-194 (1997). Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry as a Rapid Quality Control Method in Oligonucleotide Synthesis.Twenty oligonucleotides can be analyzed in less than one hour in unattended operation by taking advantage of the commonly available automation features of MALDI/MS instruments. 3-Hydroxypicolinic acid is used as a matrix, and cation-exchange resin is applied to targets with sample and matrix to diminish cation-adducts. MALDI/MS provides estimates of byproduct levels similar to capillary electrophoresis for oligonucleotides up to 35 bases.
  

Mass Spectrometry

 
• Roepstorff, P. (1997) Current Opinions in Biotechnology 8, 6-13. Mass Spectrometry in Protein Studies from Genome to Function.This article provides a brief overview of recent developments in mass spectrometry, especially as they pertain to the high-throughput analysis needed for proteome projects. The topics covered and described more fully in the comprehensive, up-to-date bibliography include: protein identification by mass fingerprinting and sequence tags, de novo sequencing, and analysis of protein-ligand interactions.
  
• Neubauer, G. and Mann, M. (1997) Journal of Mass Spectrometry 32, 94-98. Parent Ion Scans of Large Molecules.This article describes an approach that may be useful for screening proteins for post-translational modifications. During mass analysis of intact proteins, collisional dissociation can fragment them to give the same low-mass ions characteristic for post-translational modifications in peptides, e.g., phosphate ion (m/z of 79) for phosphorylation and oxonium ion (m/z of 204) for glycosylation.
  
• McCormack, A. L., Schieltz, D.M., Goode, B., Yang, S., Barnes, G., Drubin, D. and Yates, J.R. (1997) Analytical Chemistry 69, 767-776. Direct Analysis and Identification of Proteins in Mixtures by LC/MS/MS and Database Searching at the Low-Femtomole Level.This paper tests the feasibility of identifying proteins in mixtures by LC/MS/MS with automated data analysis. Artificial protein mixtures were made by mixing tryptic digests of seven proteins commonly used as SDS-PAGE molecular weight markers (2 picomoles total) with smaller amounts (40 femtomoles) of casein, playing the role of an "unknown" protein. This complex digest was separated by capillary HPLC, the eluting peptides were analyzed with an on-line MS/MS "detector", and the MS/MS data were interpreted with the SEQUEST program. The "unknown" component was successfully identified from its peptide fragments, even though it was a minor component of the mixture.
  

Amino Acid and Sequence Analysis

 
• Cladrowa-Runge, S. and Rizzi, A. (1997) Journal of Chromatography A 759, 157-165. Enantioseparation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate-derivatized-amino acids by capillary zone electrophoresis using native and substituted b-cyclodextrins as chiral additives. I. Discussion of optimum separation conditions.AQC is frequently used as a pre-column derivatization reagent in HPLC-based amino acid analysis. Here it is used in capillary electrophoresis separations in an attempt to separate the D and L isomers of all 19 naturally occurring chiral amino acids. This is accomplished simply by adding cyclodextrins cyclic carbohydrate polymer clathrates that can include organic substances within their hydrophobic cavities so the CE electrolyte. In this preliminary study, seven chiral pairs were baseline resolved, and nine others were partially resolved; a different cyclodextrin was needed to separate isomers of Arg, Lys, and His. Because the objective is to find a cyclodextrin that will resolve all 19 chiral amino acids, a companion article describing the cyclodextrin-amino acid interaction from a theoretical point of view immediately follows this one.
  
• Kawakami, T., Kamo, M., Takamoto, K., Miyazaki, K., Chow, L.-P., Ueno, Y. and Tsugita, A. (1997) Journal of Biochemistry 121, 68-76. Bond-Specific Chemical Cleavages of Peptides and Proteins with Perfluoric Acid Vapors: Novel Peptide Bond Cleavages of Glycyl-Threonine, the Amino Side of Serine Residues and the Carboxyl Side of Aspartic Acid Residues.Acids can selectively cleave the Asp-Pro bonds of peptides and proteins. This paper explores gas-phase cleavage with heptafluorobutyric acid (HFBA) and uncovers a previously undocumented cleavage of Gly-Thr bonds at yields approaching 70% (with 75% HFBA at 30-40° for 24 hours). Under certain conditions, Asp-X and X-Ser bonds are also cleaved. Acid cleavage is conducted in an apparatus similar to the commonly used Picotag workstation, and sample work-up is simple due to the volatility of HFBA.

Protein Characterization and Analysis

 
• Warner, D.L. and Dorsey, J.G. (1997) LC·GC 15, 254-262. Reduction of Total Analysis Time in Gradient Elution, Reversed-Phase Liquid Chromatography.Adding 1-propanol to each reversed-phase HPLC solvent at a final concentration of 3% significantly reduced the time needed for column re-equilibration, presumably due to propanol's effectiveness as a wetting agent in mobile phases of high aqueous content.
  
• Courchesne, P.L. and Patterson, S.D. (1997) BioTechniques 22, 244-250. Manual Microcolumn Chromatography for Sample Cleanup Before Mass Spectrometry.A simple, rapid, and effective clean-up procedure for MALDI-MS samples. "Offensive" components such as guanidine HCl, urea, glycerol, and EDTA were removed from Lys-C digests of standard proteins, using commercially available reversed-phase HPLC guard columns (0.3 x 1 mm). Clean-up was considered successful when samples gave interpretable spectra after, and not before, the procedure.
  
• Amado, F. M. L., Santant-Marques, M. G., Ferrer-Correia, A. J. and Taylor, K. B. (1997) Analytical Chemistry 69, 1102-1106. Analysis of Peptide and Protein Samples Containing Surfactants by MALDI-MS.It is generally thought that peptide and protein samples contaminated by SDS cannot be analyzed successfully by MALDI-MS without detergent removal. This paper documents an alarmingly simple fix: increase the concentration of SDS to 0.3-10%. Some samples gave signals as strong in 10% SDS as they did in 0% SDS, using a-cyano-hydroxycinnamic acid as matrix. Increasing detergent concentration sometimes works for samples containing CHAPS but does not seem to work at all for samples containing Triton X-100.
  

Sulfhydryl Analysis

 
• Wu, J. and Watson, J.T. (1997) Protein Science 6, 391-398. A novel methodology for assignment of disulfide bond pairings in proteins.Disulfide linkages in proteins are mapped by partially reducing denatured proteins with Tris(2-carboxylethyl)phosphine, cyanylating the thiol groups, HPLC purifying the peptides, and then analyzing them by MALDI-MS. When treated with base, the cyanylated peptides are cleaved amino-terminal to the cyanylated residue. After reduction, disulfide bonds can then be located by mass analysis of the cleavage products.

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