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
Takamoto, K., Kamo, M., Kubota, K., Satake, K., and Tsugita, A. (1995) European Journal of Biochemistry 228, 362-372. "Carboxy-terminal degradation of peptides using perfluoracyl anhydrides. A C-terminal sequencing method."
Continuing work by this group on the development of carboxyl-terminal sequencing procedures resulted in greater degradation with no internal peptide bond cleavage. Starting with about 1 nmol of a 23-residue synthetic peptide, the first twenty residues were successfully sequenced.
Manneberg, M., Lahm, H.-W., and Fountoulakis, M. (1995) Analytical Biochemistry 224, 122-127. "Oxidation of Cysteine and Methionine Residues during Acid Hydrolysis of Proteins in the Presence of Sodium Azide."
The commonly used bacteriostat NaN3 caused unanticipated oxidation of Cys and Met residues during standard HCl hydrolysis prior to amino acid analysis. Substituting methanesulfonic acid for HCl greatly minimized oxidation of these two residues.
Fadden, P. and Haystead, T. A. J. (1995) Analytical Biochemistry 225, 81-88. "Quantitative and Selective Labeling of Phosphoserine on Peptides and Proteins: Characterization at the Attomole Level by Capillary Electrophoresis and Laser-Induced Fluorescence."
An extremely sensitive phosphoserine labeling scheme with an overall efficiency of more than 87%. Samples are treated with 1,2-ethanedithiol and then tagged with 6-iodoacetamidofluorescein. This could allow non-radioactive detection of phosphorylation sites in vivo.
Capillary Electrophoresis
Issaq, H. J. and Chan, K. C. (1995) Electrophoresis 16, 467-480. "Separation and detection of amino acids and their enatiomers by capillary electrophoresis: A review."
Comprehensive article describing capillary electrophoresis, micellar electrokinetic chromatography, pre- and post-column derivatization reactions, types of chromophores and fluorophores, direct and indirect detection, and conditions for separation of amino acid enatiomers.
Kim, N. J., Kim, J. H., and Lee, K.-J. (1995) Electrophoresis 16, 510-515. "Application of capillary electrophoresis to amino acid sequencing of peptides."
Initial study defining the conditions (with optimization) for micellar electrokinetic chromatography of PTH amino acids following Edman degradation. Sensitivity and detection limits were not addressed in this report.
DNA Sequencing
Tabor, S. and Richardson, C. C. (1995) Proceedings of the National Academy of Sciences USA 92, 6339-6343. "A single residue in DNA polymerases of the Eschericia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxynucleotides."
Most polymerases used in DNA sequencing incorporate ddNMPs only at extremely high ddNTP/dNTP molar ratios. Active site hybrids of T7, E. coli, and T. aquaticus DNA polymerases were constructed to identify the molecular basis for this discrimination against ddNTPs. When Phe667 of Taq DNA polymerase is mutated to Tyr, discrimination against fluorescent ddNTPs drops 1000-fold compared to the wildtype enzyme, making this and other mutant enzymes more effective reagents for automated DNA sequencing.
DNA/RNA Synthesis
Lashkari, D. A., Hunicke-Smith, S. P., Norgrenm R. M., Davis, R. W., and Brennan, T. (1995) Proceedings of the National Academy of Sciences USA 92, 7912-7915. "An automated multiplex oligonucleotide synthesizer: Development of high-throughput, low- cost DNA synthesis."
Describes a new generation of 96-column DNA synthesizers that have been responsible for the decrease in cost of some commercial oligonucleotide synthesis. The instrument can make a plate of 96 different 20-base oligonucleotides at 20 nmol scale in less than 5 hours and at a cost of only 13 cents per base.
Mass Spectrometry
Blackledge, J. A. and Alexander, A. J. (1995) Analytical Chemistry 67, 843-848. "Polyethylene Membrane as a Sample Support for Direct Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of High Mass Proteins."
Additional work by these authors showing the properties and advantages of polyethylene as a MALDI sample support. Highly accurate data are obtained for large proteins, up to 200 kDa.
Patterson, S. D. (1995) Electrophoresis 16, 1104-1114. "Matrix-assisted laser-desorption/ionization mass spectrometric approaches for the identification of gel-separated proteins in the 5-50 pmol range."
A general scheme is described to obtain mass data for gel-separated proteins after transfer to Immobilon-CD membranes. About 10% of the band is directly analyzed by MALDI-MS, about 10% is digested with cyanogen bromide then mass analyzed, and the remaining sample is digested with Lys-C before mass analysis. This strategy attempts to maximize mass information for protein identification through database searches. Two of three test proteins at the 5 pmol level were successfully identified using this procedure.
Schmidt, M., Eberhard, K., Beyermann, M., and Bienert, M. (1995) Peptide Research 8, 238-242. "Instability of Side-Chain Protecting Groups During MALDI-TOF Mass Spectrometry of Peptide Fragments."
Acidic matrices such as sinapinic acid and 2,5-dihydroxybenzoic acid cause partial cleavage of acid-labile sidechain protecting groups commonly used during Fmoc peptide synthesis (e.g., Arg-Pmc/Mtr/Pbf; Lys-Boc; His-Trt; and Asp-OtBu), which could compromise data interpretation. Cleavage was not observed with neutral matrices like 2,4,6-trihydroxy-acetophenone and 2-amino-5-nitropyridine. The popular peptide matrix, [[alpha]]-cyano-4-hydroxycinnamic acid, was not investigated.
Qin, J. and Chait, B. T. (1995) Journal of the American Chemical Society 117, 5411-5412. "Preferential Fragmentation of Protonated Gas-Phase Peptide Ions Adjacent to Acidic Amino Acid Residues."
Cautionary note describing specific fragmentation at Asp and Glu residues of Arg-containing peptides (eight examples), observed in a MALDI quadropole ion trap instrument with and without precursor selection. Potential use for peptide mapping of proteins and peptides.
Hess, D., Nika, H., Chow, D. T., Bures, E. J., Morrison, H. D., and Aebersold, R. (1995) Analytical Biochemistry 224, 373-381. "Liquid Chromatography-Electrospray Ionization Mass Spectrometry of 4-(3-Pyridinylmethylaminocarboxypropyl)phenylthiohydantoins."
Description of chromatographic, sequencer, and ES-MS systems needed to perform on-line mass analysis of this novel PTH analog for amino-terminal sequencing. Detection limits are reported in the sub-pmol range. See also Bures, E. J. et al. (1995) Analytical Biochemistry 224, 364-372, for a detailed report by this group on the synthesis and properties of this reagent.
Peptides--Chemistry and Purification
Cui, H., Leon, J., Reusaet, E., and Bult, A. (1995) Journal of Chromatography A 704, 27-36. "Selective determination of peptides containing specific amino acid residues by high-performance liquid chromatography and capillary electrophoresis."
Useful compilation of HPLC and CE methods for identification of intentionally derivatized Cys, Arg, Trp, Tyr, and Pro residues in peptides. The relative merits of derivatization are discussed in terms of sensitivity, selectivity, specificity, and detectability.
Ohguro, H. and Palczewski, K. (1995) FEBS Letters 368, 452-454. "Separation of phospho- and non-phosphopeptides using reverse phase column chromatography."
Different counterions (heptafluorobutyric acid, pentafluoropropionic acid, phosphoric acid, and trifluoroacetic acid) were investigated for their effectiveness during C18RP-HPLC separation of these modified peptides using water/acetonitrile gradients. The use of heptafluoro-butyric acid provided superior separation of the target phosphopeptide from the non-phosphorylated parent species.
Breenan, T. V. and Clarke, S. (1995) International Journal of Peptide and Protein Research 45, 547-553. "Effect of adjacent histidine and cysteine residues on the spontaneous degradation of asparaginyl- and aspartyl-containing peptides."
Experimental effort using synthetic peptides to define Asn- and Asp-containing motifs subject to deamidation, isomerization, peptide bond cleavage, and racemization. The authors conclude that His-Asp sequences are prone to spontaneous degradation in peptides and proteins.
Olejnik, J., Sonar, S., Krzymañska-Olejnik, E., and Rothschild, K. J. (1995) Proceedings of the National Academy of Sciences USA 92, 7590-7594. "Photocleavable biotin derivatives: A versatile approach for the isolation of biomolecules."
The synthesis of a photocleavable biotin N-hydroxysuccinimide ester derivative is described. Applications using leucine-enkephalin as a model substrate show biotin attachment at the amino-terminus as expected, unaltered high affinity of the conjugate to avidin/streptavidin, and rapid and quantitative release of the model peptide after photolysis with ultraviolet light at 365 nm. Other potential uses such as cell specific isolations are anticipated.
Protein Characterization and Analysis
Means, G. E. and Feeney, R. E. (1995) Analytical Biochemistry 224, 1-16. "Reductive Alkyation of Proteins".
Comprehensive presentation detailing the organic chemistry used for reductive alkylation of proteins and peptides. Conditions for use of four different reducing agents are summarized as well as methods and reagents for non-biosynthetic radiolabeling.
Ségalas, I., Thai, R., Ménez, R., and Vita, C. (1995) FEBS Letters 371, 171-175. "A particularly labile Asp-Pro bond in the green mamba muscarinic toxin MTX2. Effect of protein conformation on the rate of cleavage."
Shows the unexpected result that the rate of acid cleavage (0.2 M glycine-HCl pH 1.5 to 5.0, 45[[ring]]C) at this linkage was dramatically slower for the denatured protein (plus 6 M guanidine hydrochloride) than for the native protein, probably because of an autocatalytic spatial arrangement of these residues in the native structure. Caution should be exercised for techniques employing mild acid conditions, e.g., NMR experiments, of Asp-Pro containing samples.
Fernandez-Patron, C., Madrazo, J., Hardy, E., Mendez, E., Frank, R., and Castellanos-Serra, L. (1995) Electrophoresis 16, 911-920. "Single-step electrotransfer of reversed-stained proteins from SDS-PAGE onto RP minicartridge and subsequent desalting and elution with a conventional HPLC gradient system for analysis."
A one-step high-recovery (up to 90 %) procedure for mid-pmol amounts (about 50 pmol) of proteins from 6 to 68 kDa after electrophoresis is reported. The material is isolated in volatile reverse phase solvents facilitating subsequent analysis by amino-terminal sequencing, mass spectrometry, and proteolysis. A detailed schematic of the minicartridge is provided; this apparatus can be assembled from commercially available supplies.
Davis, M. T., Lee, T. D., Ronk, M., and Hefta, S. A. (1995) Analytical Biochemistry 224, 235-244. "Microscale Immobilized Protease Reactor Columns for Peptide Mapping by Liquid Chromatography/Mass Spectral Analyses."
Details on the preparation and use of an immobilized trypsin reactor followed by LC/MS analysis. A sensitivity of 10 pmol is demonstrated, and protein genetic variants can be analyzed in less than two hours.
Samuel, M. (1995) Analytical Biochemistry 224, 457-459. "PHAST 2D, a Two-Dimensional Electrophoretic Technique on a Single Gel under Native and Denaturing Conditions Using Pharmacia PhastSystem."
Simple but rapid and effective means of evaluating protein-protein interactions, e.g., oligomeric states, by two-dimensional native and SDS electrophoresis on ug quantities of protein.
Sulfhydryl Analysis
Ming, D., Markley, J. L., and Hellekant, G. (1995) Biotechniques 18, 808-810. "Quantitation of Cysteinyl Sulfhydryl Residues in Peptides and Proteins by ESI-MS or MALDI-MS."
Pyridylethylation of proteins and peptides followed by mass analysis provided a means of accurately quantitating sulfhydryl groups. It should be possible to determine the number of free and disulfide-linked thiols by this procedure.
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