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
Bailey, J. (1995) Journal of Chromatography A 705, 47-65. "Chemical methods of protein sequence analysis."
Review, with a chemical flavor, containing 117 references (through mid-1994) on protein sequencing methodology. Supports, chemistries (coupling, cleavage, and conversion), and detection for both amino- and carboxyl-terminal sequencing are described.
Tempst, P., Geromanos, S., Elicone, C., and Erdjument-Bromage, H. (1994) METHODS: A Companion to Methods in Enzymology 6, 248-261. "Improvements in Microsequencer Performance for Low Picomole Sequence Analysis."
A five-year update of a previous review by this group on how to successfully perform low-level sequencing. Detailed suggestions with supporting data on key areas including sample preparation and handling, instrument and chromatography optimization, and uncompromising quality assurance protocols. Realistic expectations for users of an "average" sequencing facility are discussed.
Chang, N. (1995) International Journal of Peptide and Protein Research 45, 157-163. "Role of Peptide Hydrophilicity on Determination of Microsequencing Efficiency."
Addresses the problem of sequencing relatively short peptides by comparing the sequencing efficiency of one peptide on PVDF-P, PVDF-PB, PVDF-N, and chemically modified glass-fiber filters. With PVDF-P and PVDF-PB, polybrene treatment gave higher repetitive yields. Sequencing became less efficient with lower peptide hydrophilicity, as calculated by the Kyte-Doolittle method.
Capillary Electrophoresis
Strege, M. A. and Lagu, A. L. (1995) Electrophoresis 16, 642-646. "Capillary electrophoretic separations of biotechnology-derived proteins in E. coli fermentation broth."
Procedures are described and validated for quantitative monitoring of recombinant proteins in fermentation broths by capillary electrophoresis. Addition of magnesium sulfate and acetonitrile to the separation buffer improved selectivity and efficiency in a 50 um polyacrylamide-coated capillary. Run times of 30 minutes at 25 kV for a 57 cm capillary are reported with relative standard deviations of about 6% for peak quantitation.
Adamson, N. J. and Reynolds, E. C. (1995) Electrophoresis 16, 525-528. "High performance capillary electrophoresis of casein phosphopeptides containing 2-5 phosphoseryl residues: Relationship between absolute electrophoretic mobility and peptide charge and size."
Empirical relationships relating charge, size, and mobility are derived for mono-, di-, and tri-phosphorylated peptides. Data should be applicable to capillary electrophoresis of phosphopeptides in general.
Tadey, T. and Purdy, W. C. (1995) Electrophoresis 16, 574-579. "Capillary electrophoretic resolution of phosphorylated peptide isomers using micellar solutions and coated capillaries."
Another approach to analyze phosphopeptides by capillary electrophoresis. SDS in a buffered pH 6.1 solution efficiently resolved three synthetic tyrosylphosphopeptides derived from the insulin receptor. Coating the capillary reduced sample adsorption but also decreased resolution in some instances.
Carbohydrate Structure and Analysis
Reinhold, V. N., Reinhold, B. B., and Costello, C. E. (1995) Analytical Chemistry 67, 1772-1784. "Carbohydrate Molecular Weight Profiling, Sequence, Linkage, and Branching Data: ES-MS and CID."
A "perspective" article describing the value of mass spectrometry for analysis and structure determination of this important class of biomolecules. Written in such a way that the non-specialist will gain an appreciation for the complexity and diversity of carbohydrate structure.
DNA Sequencing
Borman, S. (1995) Chemical and Engineering News 23 (30), 37-39. "Developers of novel DNA sequencers claim major performance advances."
Two sequencing strategies under development are described: a multichannel (100-1,000) capillary electrophoresis technique and a hybridization "superchip". An intriguing glimpse into the technological future, but with sparse technical descriptions.
Manabe, T., Chen, N., Terabe, S., Yohda, M., and Endo, I. (1994) Analytical Chemistry 66, 4243-4252. "Effects of linear polyacrylamide concentrations and applied voltages on the separation of oligonucleotides and DNA sequencing fragments by capillary electrophoresis."
Describes modifications to a commercial instrument that allow single-base separations of oligonucleotides as long as 500 bases, a resolution suitable for DNA sequencing. Key modifications include bonding a layer of polyacrylamide to the inner surface, using a linear polyacrylamide core, and not coiling the capillary.
DNA/RNA Synthesis
Temsamini, J. Kubert, M., Agrawal, S. (1995) Nucleic Acids Research 23 (11), 1841-1844. "Sequence Identity of the n-1 Product of a Synthetic Oligonucleotide."
Analysis of the n-1 products of a 25-base oligonucleotide shows a mixed population of 24mers. Truncations at the 5' end were not detected, but truncations at the 3' end accounted for a large part of the n-1 products, probably from synthesis at uncapped sites on the CPG support and inefficient synthesis near the 3' end.
Sproat, B., Colonna, F., Mullah, B., Tsou, D., Andrus, A., Hampel, A., and Vinayak, R. (1995) Nucleosides and Nucleotides 14, 255-273. "An efficient method for the isolation and purification of oligoribonucleotides."
Review of advances in the synthesis, deprotection, and purification of synthetic RNA, presented in a readable, techniques-oriented manner. A "must have" for experienced RNA chemists.
Mass Spectrometry
Borman, S. (1995) Chemical and Engineering News 73(25), 23-31. "Biochemical Applications of Mass Spectrometry Take Flight."
Readable, historic review of mass analysis applications to biological problems culminating with up-to-date examples of MALDI-TOF and electrospray mass spectrometry.
Nguyen, D. N., Becker, G. W., and Riggen, R. M. (1995) Journal of Chromatography A 705, 21-45. "Protein Mass Spectrometry: Applications to Analytical Biotechnology."
Comprehensive discussion of "what can go wrong" with recombinant proteins in biopharmaceutical research and development and the use of mass spectrometry as an analytical tool to resolve these conflicts. Several examples are presented under the headings: molecular mass determination, peptide mapping/MS, sequencing, non-covalent interactions, and protein folding/higher order structural characterization.
Bai, J., Qian, M. G., Liu, Y., Liang, X., and Lubman, D. M. (1995) Analytical Chemistry 67, 1705-1710. "Peptide Mapping by CNBr Degradation on a Nitrocellulose Membrane with Analysis by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry."
Nitrocellulose-bound proteins are digested in situ with CNBr and mass analyzed by MALDI. Better signals are obtained if the fragments are eluted from the membrane first with acetonitrile/water or propanol/water mixtures, with sensitivity in the pmol range. Overall, a rapid mapping strategy.
Murphy, C. M. and Fenselau, C. (1995) Analytical Chemistry 67, 1644-1645. "Recognition of the Carboxy-Terminal Peptide in Cyanogen Bromide Digests of Proteins."
After CNBr digestion, HPLC-purified peptides are esterified with acidic methanol and mass analyzed, to distinguish carboxyl-terminal peptides from internal peptides. Carboxyl-terminal peptides show mass gains that are multiples of 14 Da (from methylation of sidechain and [[alpha]]-carboxylate groups), but lactones at carboxyl-termini of internal peptides react to produce hydroxyl methyl esters and 32 Da mass gains.
Karas, M., Bahr, U., Strupat, K., Hillenkamp, F,., Tsarbopoulos, A., and Pramanik, B. N. (1995) Analytical Chemistry 67, 675-679. "Matrix Dependence of Metastable Fragmentation of Glycoproteins in MALDI TOF Mass Spectrometry."
Systematic study showing the effect of different matrices on metastable decay during reflectron MALDI-TOF mass analysis. Glycoproteins could be analyzed with high resolution and without fragmentation with the matrix 3-hydroxypicolinic acid, commonly used for oligonucleotide desorption.
Peptides--Chemistry and Purification
Tamamura, H., Otaka, A., Nakamura, J., Okubo, K., Koide, T., Ikeda, K., Ibuka, T., and Fujii, N. (1995) International Journal of Peptide and Protein Research 45, 312-319. "Disulfide bond-forming reaction using a dimethyl sulfoxide/aqueous HCl system and its application to regioselective two disulfide bond formation."
Sequential treatment of acetamidomethyl-cysteine peptides with silver trimethanesulfonate and DMSO/HCl resulted in quantitative conversion of the protected cysteine into cystine with little or no side reactions for Met, Tyr, and Trp. This two-step procedure combined with air oxidation allowed regioselective disulfide bond formation.
Hunter, M. J. and Komives, E. A. (1995) Analytical Biochemistry 228, 173-177. "Deprotection of S-Acetamidomethyl Cysteine-Containing Peptides by Silver Trifluoromethanesulfonate Avoids the Oxidation of Methionines."
A procedure to produce a 40-residue peptide containing three disulfide bonds using S-trityl and S-acetamidomethyl protecting groups. The peptide was obtained with good yield by deprotection with silver trifluoromethanesulfonate and oxidation with DMSO/HCl, with minimal oxidation of methionine residues.
Ball, H. L., Bertolini, G., Levi, S., and Mascagni, P. (1994) Journal of Chromatography A 686, 73-83. "Purification of synthetic peptides with the aid of reversible chromatographic probes."
Two peptides, one made using Fmoc chemistry (46 residues) and another using tBoc chemistry (104 residues), were tagged with base-labile reversible probes prior to resin cleavage. The probes--two lipophilic derivatives of 9-(hydroxymethyl)fluorene-4-carboxylic acid and one biotin analog of this--allowed facile isolation of the full-length peptides and were easily removed after HPLC purification by base hydrolysis.
Protein Characterization and Analysis
Schöneich, C., Hühmer, A., Rabel, S., Stobaugh, J., Jois, S., Larive, C., Siahaan, T., Squier, T., Bigelow, D., and Williams, T. (1995) Analytical Chemistry 67, 155R-181R. "Separation and Analysis of Peptides and Proteins."
Up-to-date (1993-1994 articles) review on the use of analytical and preparative HPLC, CE, NMR, IR, CD, fluorescence, and MS to fractionate and identify various peptide and protein components. Bibliography nicely separates each topic, allowing easy access to references.
Hellman, U., Wernstedt, C., Góñez, J., and Heldin, C.-H. (1995) Analytical Biochemistry 224, 451-455. "Improvement of an "In-Gel" Digestion Procedure for the Micropreparation of Internal Protein Fragments for Amino Acid Sequencing."
Recipes and tips are given to improve peptide yields when using this popular in situ proteolytic digestion method. Completely drying gel pieces increased peptide recovery about two-fold.
Jenö, P., Mini, T., Moes, S., Hintermann, E., and Horst, M. (1995) Analytical Biochemistry 224, 75-82. "Internal Sequences from Proteins Digested in Polyacrylamide Gels."
A protocol for "in-gel" Lys-C digestion of reduced and alkylated Coomassie-stained proteins. Cleavages faithfully mimic the corresponding solution digests by producing the same number of peptides and similar yields. Internal sequence data can be obtained from low ug amounts of two-dimensional gel samples.
Gianazza, E. (1995) Journal of Chromatography A 705, 67-87. "Isoelectric focusing as a tool for the investigation of post-translational processing and chemical modifications of proteins."
The use and interpretation of immobilized pH gradients in isoelectric focusing gel electrophoresis is described in this review. Particular emphasis is given to the identification of modified (post-translational and chemical) proteins by electrophoretic mobility and quantitative scanning.
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