Article Watch

Amino Acid and Sequence Analysis

• Gorbics, L., Urge, L. and Otvos, L. Jr. (1994) Journal of Chromatography A 676, 169-176. Comparative and optimized dabsyl-amino acid analysis of synthetic phosphopeptides and glycopeptides.
  Derivatization, hydrolysis, and RP-HPLC parameters are studied to provide an optimal protocol for the determination of these two important classes of modified amino acids at picomole sensitivity.
• Liu, H. J. (1994) Journal of Chromatography A 670, 59-66. Determination of amino acids by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and high-performance liquid chromatography with ultraviolet detection.
  This report gives the mobile phase and gradient modifications necessary for 248 nm UV detection. Sensitivity for the AQC-amino acids is comparable to PTC-amino acids.

Capillary Electrophoresis

• Deyl, Z., Tagiaro, F. and Miksík, I. (1994) Journal of Chromatography B 656, 3-27. Biomedical applications of capillary electrophoresis.
  Detailed, current review covering all aspects of the use of capillary electrophoresis in biomedical sciences.
• Knox, J. H. (1994) Journal of Chromatography A 680, 3-13. Terminology and nomenclature in capillary electroseparation systems.
  Thorough physical treatment of CES by one of the experts in separation methodology.
• Castagnola, M., Cassiano, L., Messana, I., Nocca, G., Rabino, R., Rossetti, D. V. and Giardina, B. (1994) Journal of Chromatography B 656, 87-97. Capillary zone electrophoresis of peptides: prediction of the electrophoretic mobility and resolution.
  Further attempt at defining empirical equations extracted from CE data to describe the electrophoretic properties of known standard peptides.
• Cifuentes, A. and Poppe, H. (1994) Journal of Chromatography A 680, 321-340. Simulation and optimization of peptide separation by capillary electrophoresis.
  Computer simulation in concert with experimental data is used to derive a peptide electrophoretic mobility (m) relationship between the charge (q) and molecular mass (M). This information is then applied to interpret "real" electropherograms.
• Greve, K. F., Nashabeh, W. and Karger, B. L. (1994) Journal of Chromatography A 680, 15-24. Use of zwitterionic detergents for the separation of closely related peptides by capillary electrophoresis.
  Organic solvents and zwitterionic detergents are used to effect the micellar electrokinetic chromatographic separation of closely related analytes.

Mass Spectrometry

• Fang, L., Zhang, R., Williams, E. R. and Zare, R. N. (1994) Analytical Chemistry 66, 3696-3701. On-Line Time-of-Flight Mass Spectrometric Analysis of Peptides Separated by Capillary Electrophoresis.
  Successful interface and on-line analysis of CE and MALDI-TOFMS at high theoretical plates and mid-fmol component detection limit. Several peptide separations are shown.
• Vorm, O., Roepstorff, P. and Mann, M. (1994) Analytical Chemistry 66, 3281-3287. Improved Resolution and Very High Sensitivity in MALDI TOF of Matrix Surfaces Made by Fast Evaporation.
  Improvements in sample preparation procedures leads to increased sensitivity, easier removal of salts and impurities, faster data acquisition, and a more linear mass scale.
• Vestling, M. M. and Fenselau, C. (1994) Analytical Chemistry 66, 471-477. Poly(vinylidene diflouride) Membranes as the Interface between Laser Desorption Mass Spectrometry, Gel Electrophoresis, and in Situ Proteolysis.
  Demonstration at pmol sensitivity on the suitability of PVDF for MALDI-TOFMS analysis. Enzymatic in situ digestion protocol on the membrane provides for rapid peptide mapping analysis.
• Strupat, K., Karas, M., Hillenkamp, F., Eckerskorn, C. and Lottspeich, F. (1994) Analytical Chemistry 66, 464-470. Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Proteins Electroblotted after Polyacrylamide Gel Electrophoresis.
  Systematic study of commercial PVDF membranes and instrument parameters on the suitability of this popular support for MALDI-TOFMS analysis. Infrared radiation and a succinic acid matrix were found by these authors to be superior to other conditions tested.
• Krishnamurthy, T., Hauer, C. R., Prabhakaran, M., Freedy, J. G. and Hayashi, K. (1994) Biological Mass Spectrometry 23, 719-726. Identification of Disulfide Bridges in a Cardiotoxic Peptide by Electrospray Ionization.
  Assignment of disulfide pairings using an interrupted sequencing procedure, involving desorption of a sample from a PVDF membrane following a limited number of Edman cycles and ESI-MS analysis of the truncated sample.
• Patterson, S. D. and Katta, V. (1994) Analytical Chemistry 66, 3727-3732. Prompt Fragmentation of Disulfide-Linked Peptides during Matrix-Assisted Laser Desorption Ionization Mass Spectrometry.
  Demonstration that linear mode MALDI-MS of disulfide-linked peptides yields both intact parent ion and constituent monomer peptide ions. Extremely useful for rapid screening of HPLC fractions to identify candidate disulfide-linked fragments requiring further analysis.
• Crimmins, D. L., Saylor, M., Rush, J., and Thoma, R. S. (1995) Analytical Biochemistry 226, 355-361. Facile, In Situ Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Analysis and Assignment of Disulfide Pairings in Heteropeptide Molecules.
  Di-, tri- and tetrapeptides linked by single disulfide bonds partially decompose during linear MALDI mass analysis in a manner that allows assignment of peptide-to-peptide linkages. A single analysis of pmol quantities of peptide complexes provides mass and structural information about the intact complex and its components.
• Papac, D. I., Hoyes, J. and Tomer, K. B. (1994) Protein Science 3, 1485-1492. Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.
  Rapid, high sensitivity procedure to identify linear epitopes in antigens. Should be useful for the study of other macromolecule-macromolecule interactions as well.
• Zhao, Y. and Chait, B. T. (1994) Analytical Chemistry 66, 3723-3726. Protein Epitope Mapping By Mass Spectrometry.
  Three-step rapid screening procedure to define antigenic linear epitopes requiring only pmol sample amounts.

Peptides-Chemistry and Purification

• Hohsaka, T., Sato, K., Sisido, M., Takai, K. and Yokoyama, S. (1994) FEBS Letters 344, 171-174. Site-specific incorporation of photofunctional non-natural amino acids into a polypeptide through in vitro protein synthesis.
  An E. coli system coupled with chemically misacylated tRNACCU allows efficient incorporation of photoactive modified amino acids into proteins.

Protein Characterization and Analysis

• Bonaventura, C., Bonaventura, J., Stevens, R. and Millington, D. (1994) Analytical Biochemistry 222, 44-48. Acrylamide in Polyacrylamide Gels Can Modify Proteins during Electrophoresis.
  Warning, backed-up by MS data, that acrylamide can covalently modify proteins during electrophoresis. Some reaction parameters are investigated.
• Patterson, S. D. (1994) Analytical Biochemistry 221, 1-15. From Electrophoretically Separated Proteins to Identification: Strategies for Sequence and Mass Analysis.
  First-rate review on protein electrophoresis methodologies as a fractionation process to provide suitable samples for mass analysis or sequencing with the ultimate goal of sample identification.
• Nokaihara, K., Kuriki, T. and Morita, N. (1994) Journal of Chromatography A 676, 233-238. Two-dimensional electrophoresis as a complementary method of isolating peptide fragments of cleaved proteins for internal sequencing.
  This paper shows that classical 2D IEF/MW gels can successfully isolate proteolyzed protein samples in instances where HPLC cannot. Sequence data on the electroblotted fragments are given.
• Bermudez, A., Daban, J.-R., Garcia, J. R. and Mendez, E. (1994) Biotechniques 16, 621-624. Direct Blotting, Sequencing and Immunodetection of Proteins after Five-Minute Staining of SDS and SDS-Treated IEF Gels with Nile Red.
  Nile Red fluorescently stains electrophoresed proteins which can then be blotted to PVDF for further analysis, e.g., Western blotting or N-terminal sequencing.
• Elliott, J. I., Stone, K. L., and Williams, K. R. (1993) Analytical Biochemistry 211, 94-101. Synthesis and Use of an Internal Amino Acid Sequencing Standard Peptide.
  Design, synthesis, and use of an internal sequencing standard containing norleucine and succinyl-lysine is described. The standard permits repetitive yield and carryover calculations during each sequencing run and helps early detection of suboptimal instrument performance.
• Parmelee, D. C., Hoang, T. N., Benjamin, T. and Sechi, S. (1994) Analytical Biochemistry 219, 71-81. Noninterfering Synthetic Peptides as Internal Controls for Amino Acid Sequencing of Sample Unknowns.
  Rational design and detailed characterization of internal sequencing standards is described. Allows sequencer, chromatography, and data analysis systems verification.

Sulfhydryl Analysis

• Singh, R. and Whitesides, G. M. (1994) Bioorganic Chemistry 22, 109-115. Reagents for Rapid Reduction of Native Disulfide Bonds in Proteins.
  Three disulfide reductants were evaluated in terms of kinetics and solvent composition regarding disulfide bond cleavage in several native proteins. Bis(2-mercaptoethyl)sulfone is more effective than the familiar dithiothreitol.
• Ramseier, U. and Chang, J.-Y. (1994) Analytical Biochemistry 221, 231-233. Modification of Cysteine Residues with N-Methyl Iodoacetamide. The modification of cysteine residues with an iodoacetamide analog is described. Chromatographic conditions to identify the PTH derivative found during sequence analysis and the DABSYL derivative found during amino analysis are given.
• Yamada, H., Seno, M., Kobayashi, A., Moriyama, T., Kosaka, M., Ito, Y. and Imoto, T. (1994) Journal of Biochemistry (Tokyo) 116, 852-857. An S-Alkylating Reagent with Positive Charges as an Efficient Solubilizer of Denatured Disulfide-Containing Proteins.
  The synthesis and characterization of a novel bis quaternary ammonium bromide reagent for the modification of cysteine residues is presented. Modified cysteines carry a +2 positive charge so fragments containing these derivatives are highly soluble and chromatographically easy to isolate.

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