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.
Amino Acid and Sequence Analysis
Crimmins, D.L. and Cherian, R. (1997) Analytical Biochemistry 244, 407-410. Increasing the Sensitivity of 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate Amino Acid Analysis: A Simple Solution.
AQC derivitization uses a simple, commercially available "shake and bake" reagent. Unfortunately only 20% of the derivatized sample can be HPLC analyzed, because the usual protocol leaves derivatized amino acids in 20% acetonitrile. In this paper it is shown that if the samples are dried and redissolved in 5% acetonitrile, 80% of the sample can be injected for HPLC analysis, without compromising early eluting amino acid derivatives. Hydrolyzing, derivatizing, drying, redissolving, and injecting the sample in a single HPLC autosampler vial minimizes sample handling for high recoveries, despite the additional steps.
Bondi, R.M., Walz, K., Issinger, O.-G., Engel, M. and Passeron, S. (1996) Analytical Biochemistry 242, 165-171. Discrimination between Acid and Alkali-Labile Phosphorylated Residues on Immobilon: Phosphorylation Studies of Nucleoside Diphosphate Kinase.
A straightforward procedure for distinguishing proteins containing phosphohistidine from those containing phosphoserine or phosphothreonine, due to differences in stability after exposure to acid or base. Proteins containing radioactive phosphate are electrophoresed and blotted to PVDF membranes, which are then sequentially treated with pH 1 and with pH 14 solutions. Loss of radioactivity at pH 1 indicates pHis (or pTyr), and at pH 14 pSer/pThr.
Hwang, B.J., Smith, A.J. and Chu, G. (1996) Journal of Chromatography B 686, 165- 175. Internal Sequence Analysis of Proteins Eluted from Polyacrylamide Gels.
Sequence analysis of high molecular weight proteins can be difficult because of poor transfer to membrane supports. Here an alternate strategy based on "elution-digestion" was used to prepare peptides from 45-200 kDa proteins. After elution from gels in the presence of SDS and Lys C, samples were extracted with heptane-isoamyl alcohol to remove SDS and then digested further in solution before fractionation by HPLC. Interestingly, organic extraction gave higher sample recoveries than typically found for anion-exchange methods for SDS removal, and in one instance sequence was obtained from 10 pmol of a 93 kDa protein.
Capillary Electrophoresis
Bergman, A.-C. and Bergman, T. (1996) FEBS Letters 397, 45-49. Micropreparation of Peptides by Capillary Electrophoresis for Matrix Assisted Laser Desorption Mass Spectrometry.
A practical difficulty in off-line coupling of capillary
electrophoresis
and mass spectrometry is that the electrolytes favored for CE can
suppress sample ionization during mass analysis. In this report,
signal suppression caused by sodium phosphate buffers commonly used
during CE was overcome by collecting fractions into citrate, TFA, or
HCl prior to mass analysis. When HCl was used, good spectra were
obtained from fmol amounts of material fractionated in the presence
of sodium phosphate, without sodium adduct artifacts.
CarbohydratesStructure and Analysis
Kuster, B., Naven, T.J.P. and Harvey, D.J. (1996) Journal of Mass Spectrometry 31, 1131-1140. Rapid Approach for Sequencing Neutral Oligosaccharides by Exoglycosidase Digestion and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.
Low-pmol amounts of neutral, underivatized oligosaccharides are characterized by MALDI and are then recovered from the target, sequentially digested with exoglycosidases, and re-analyzed. Compared to traditional oligosaccharide sequencing approaches, this approach is quick, sidesteps derivatization, uses small reaction volumes so substrate concentrations are nearer enzyme Km's, and can be sensitive and efficient due to sample re-use.
DNA Sequencing and Analysis
Goffeau, A., Barrell, B.G., Bussey, H., Davis, R.W., Dujon, B., Feldmann, H., Galibert, F., Hoheisel, J.D., Jacq, C., Johnston, M., Louis, E.J., Mewes, H.W., Murakami, Y., Philippsen, P., Tettelin, H. and Oliver, S.G. (1996) Science 274, 546-567. Life with 6000 Genes.
A brief, post-genome overview of the prospects for yeast biology after completing the yeast genomic sequence. Especially interesting is this group's commentary on the potential of proteome analysis, the need to maintain focus to complete other genome projects, and the relative merits of "factory" and "network" approaches to genomic sequencing.
Internet Methods
Griffin, H. (1996) Analytical Biochemistry 239, 120-122. Methods on the Internet: A Selection of Molecular Biology Protocols.
Short summaries of 11 WWW homepages that contain compilations of various methods, including some with methods forums, links to on-line journals, alphabetical directories of common laboratory chemicals, large-scale DNA sequencing protocols, and sections on keeping laboratory notebooks. These 11 homepages are listed at the URL address: http://192.215.52.3/www/journal/ab/abli.htm.
Mass Spectrometry
Jonscher, K.R. and Yates, J.R. (1997) Analytical Biochemistry 244, 1-15. The Quadrupole Ion Trap Mass SpectrometerA Small Solution to a Big Challenge.
Overview of developments in the application of ion trap mass spectrometry to biological problems, expanded and with more detail than the review written by this group for a recent issue of ABRF News.
Solouki, T., Pasa-Tolic, L., Jackson, G.S., Guan, S. and Marshall, A.G. (1996) Analytical Chenistry 68, 3718-3725. High-Resolution Multistage MS, MS2, and MS3 Matrix-Assisted Laser Desorption/Ionization FT-ICR Mass Spectra of Peptides from a Single Laser Shot.
Up to three dimensions of mass spectrometry are used to analyze tryptic digests of cytochrome c, with subfemtomole levels of detection. Digests are desalted and concentrated by step elution from SepPack reversed-phase cartridges and then ionized by MALDI in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Multi-dimensional analysis is accomplished by an ion remeasurement technique that consists of sustained off-resonance irradiation (SORI) to dissociate ions, ion isolation by stored waveform inverse Fourier transform (SWIFT), and ion axialization.
Glocker, M.O., Bauer, S.H.J., Kast, J., Volz, J. and Przybylski, M. (1996) Journal of Mass Spectrometry 31, 1221-1227. Characterization of Specific Noncovalent Protein Complexes by UV Matrix-Assisted Laser Desorption Ionization Mass Spectrometry.
Dissolving the matrix 6-aza-2-thiothymine (ATT) in aqueous solutions, without the usual organic co-solvents, enables analysis of non-covalent complexes by MALDI. Examples of the non-covalent complexes observed include dimers of leucine zipper peptides and the RNAse S peptide-protein complex. Furthermore, unlike other common matrices such as a-cyano-hydroxycinnamic acid, ATT does not interfere with on-target digestion with trypsin.
Vissres, J.P.C., Hulst, W.P., Chervet, J.-P., Snijders, H.M.J. and Cramers, C.A. (1996) Journal of Chromatography B 686, 119-128. Automated On-Line Ionic Detergent Removal from Minute Protein/Peptide Samples Prior to Liquid Chromatography-Electrospray Mass Spectrometry.
A fully automated, tandem HPLC system for LC-MS of SDS-containing samples is described. The instrument configuration is the generic one, modified by the addition of two upstream guard columnsone containing anion-exchange resin for detergent removal and the other reversed-phase matrix for sample concentration.
Xiangyu, J., Smith, J.B. and Abraham, E.C. (1996) Journal of Mass Spectrometry 31, 1309-1310. Identification of a MS-MS Fragment Diagnostic for Methionine Sulfoxide.
It is not straightforward to positively identify oxidized methionine, whether naturally occurring, intentionally produced, or the result of sample manipulation. Its presence is often inferred from changes in chromatographic elution times, tryptic peptide mapping, or masses 16 Da greater than expected. In this report, an MS-MS b-series ion with a mass 64 Da less than expected is attributed to neutral loss of methanesulfonic acid, providing a unique fingerprint for methionine sulfoxide.
PeptidesSynthesis and Purification
Botti, P., Pallin, T.D. and Tam, J.P. (1996) Journal of the American Chemical Society 118, 10018-10024. Cyclic Peptides from Linear Unprotected Peptide Precursors Through Thiazolidine Formation.
A method for producing cyclic peptides from linear, unprotected precursors is described. Peptides are synthesized with Cys at the amino-terminus and with a 1,2-amino alcohol or a 1,2-diol reactive site at the carboxyl terminus or at a sidechain. When the peptides are treated with sodium periodate, the reactive sites are converted to aldehydes, which can then react with the aminothiol functionality from amino-terminal Cys to form thiazolidines. Peptides containing 5 to 26 residues could be cyclized at concentrations as high as 20 mM without polymerization.
Delforge, D., Art, M., Gillon, B., Dieu, M., Delaive, E., Raes, M. and Remacle, J. (1996) Analytical Biochemistry 242, 180-186. Automated Solid-Phase Synthesis of Cyclic Peptides Bearing a Side-Chain Tail Designed for Subsequent Chemical Grafting.
In this scheme, synthetic peptides are designed so the carboxyl-terminal sequences are: allyl-protected Asp or Glu + aminohexanoic acid + Cys. After removal of the allyl groups from peptide-resins with metallic palladium, addition of standard coupling reagents causes the a-amino groups to react with the deprotected sidechain carboxylate group. The carboxyl-terminal cysteine residue attached to a 6-carbon spacer arm can then be used to chemically graft the cyclized peptide to another molecule.
Lukszo, J., Patterson, D., Albericio, F. and Kates, S.A. (1996) Letters in Peptide Science 3, 157-166. 3-(1-Piperidinyl)alanine formation during the preparation of C-terminal cysteine peptides with the Fmoc/t-Bu strategy.
Fmoc synthetic peptides containing carboxyl-terminal Cys are prone to a side-reaction where Cys is converted to 3-(1-piperidinyl)alanine. This side-reaction adds 51 Da to the peptide's expected mass. The by-product accumulates during the repeated exposure to piperidine needed for synthesis of long peptides, but the extent of the side-reaction varies with the resins and other reagents used during synthesis.
Schultz, J.S. and Schultz, J.S. (1996) Biotechnology Progress 12, 729-743. The Combinatorial Library: A Multifunctional Resource.
Up-to-date comprehensive review summarizing the multifaceted nature of combinatorial librariesparallel synthesis of hundreds to millions of compoundsin biomedical research, particularly as applied to the discovery mode of new compounds.
Protein Characterization and Analysis
Lui, M., Tempst, P. and Erdjument-Bromage, H. (1996) Analytical Biochemistry 241, 156-166. Methodical Analysis of Protein-Nitrocellulose Interactions to Design a Refined Digestion Protocol.
This work systematically investigates the ability of 37 organic solvents, organic bases, and detergents to elute electroblotted proteins from nitrocellulose, aimed at finding more efficient methods for in situ proteolysis. A simple, effective, one-step procedure is described based on the seldom used Zwittergent 3-16. With a 1% solution of this detergent in 100 mM ammonium bicarbonate, recoveries of some standard proteins after digestion approached 90%. This digest buffer is compatible with downstream analytical procedures, including MALDI-MS after a simple cleanup procedure.
Goldring, J.P.D. and Ravaioli, L. (1996) Analytical Biochemistry 242, 197-201. Solubilization of Protein-Dye Complexes on Nitrocellulose to Quantify Proteins Spectrophotometrically.
A quantitation procedure for proteins bound to nitrocellulose, through either direct absorption or electrotransfer after SDS-PAGE. Amido black, Ponceau S, colloidal silver, and Coomassie blue were evaluated: colloidal silver gave a linear response for 0.2 to 0.8 mg of protein and the other stains gave linear responses with 2 to 30 mg protein. The method is based on solubilizing membranes with dimethyl sulfoxide prior to spectrophotometric analysis. Although this quantitative method is destructive, it can be useful at the final stages of an experiment.