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.

Amino Acid and Sequence Analyses

van Wandelen, C. and Cohen, S. A. (1997) Journal of Chromatography A 763, 11-22. Using quaternary high-performance liquid chromatography eluent systems for separating 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate-derivatized amino acid mixtures.

Usually it is not possible to resolve standard and non-standard amino acids in a single chromatographic analysis without resorting to lengthy gradients. However, this paper describes a rapid method based on AQC derivatization of amino acids and HPLC analysis with a four-solvent system consisting of low pH buffer (pH of about 5), high pH buffer (about 6.8), acetonitrile, and water. With this method the sixteen amino acids found in Pierce standard H as well as Asn, Gln, Trp, gamma-amino butyric acid, ornithine, taurine, hydroxyproline, and hydroxylysine can be analyzed in less than one hour.

Thiede, B., Salinkow, J. and Wittman-Liebold, B. (1997) European Journal of Biochemistry 244, 750-754. C-terminal ladder sequencing by an approach combining chemical degradation with analysis by matrix-assisted laser desorption/ionization mass spectrometry.

A one-step, chemical approach for carboxyl-terminal "ladder" sequencing applicable to low-pmol quantities of peptides. The carboxyl-terminus of a peptide is activated by formation of a mixed anhydride, which cyclizes and cleaves to give a 2-thiohydantoin residue and peptide shortened at the carboxyl- terminus; in a single reaction, one addition of sequencing reagents can remove several residues from the carboxyl-terminus. The shortened peptide sequencing products are then analyzed by MALDI-MS to deduce the peptide's carboxyl-terminal sequence. The procedure was used with seventeen peptides, and the best result allowed assignment of eight residues. For some peptides, longer sequences can be assigned by treating the sample with carboxypeptidase P before chemical degradation.

Bonetto, V., Bergman, A.-C., Jornvall, H. and Sillard, R. (1997) Analytical Chemistry 69, 1315-1319. C-Terminal Sequence Analysis of Peptides and Proteins Using Carboxypeptidases and Mass Spectrometry after Derivatization of Lys and Cys Residues.

Enzymatic approaches for carboxyl-terminal "ladder" sequencing currently have two weak points: the commonly used carboxypeptidases Y and P often stop digesting samples when they encounter Cys or its derivatives (cysteic acid and carboxymethylcysteine), and it is difficult to distinguish Lys and Gln during mass analysis because of their similar masses (128.18 and 128.13 respectively). Both issues can be addressed by chemically modifying samples before carboxypeptidase treatment. Trimethylaminoethylation with (2-bromoethyl) trimethylammonium bromide converts cysteine residues to 4-thialaminine residues, which are efficiently cleaved by carboxypeptidases, and guanidination with O-methylisourea derivatizes lysine residues, allowing Lys and Gln to be confidently assigned without extremely high accuracy during mass analysis.

Capillary Electrophoresis

Yoo, Y. S., Han, Y. S., Suh, M. J. and Park, J. (1997) Journal of Chromatography A 763, 285-293. Analysis of phosphopeptides by capillary electrophoresis and matrix-assisted laser-desoprtion ionization-time-of-flight mass spectrometry.

CE and MALDI-MS are coupled "off line" to analyze phosphopeptides and their non-phosphorylated counterparts. Successful analysis requires optimizing the concentration, pH, and components of the electrophoresis buffer, and low-nmol quantities of peptides can be detected during CE by UV absorbance.

Figeys, D., Ducret, A. and Aebersold, R. (1997) Journal of Chromatography A 763, 295-306. Identification of proteins by capillary electrophoresis-tandem mass spectrometry. Evaluation of an on-line solid-phase extraction device.

Prior to CE-MS/MS analysis, dilute analytes are concentrated on-line by a solid-phase device consisting of C-18 reversed-phase support. With a tryptic digest of BSA, the limit-of-detection during CE was 33 to 300 amol/ml, with MS/MS detection limits of 660 amol to 6 fmol. Details for constructing the on-line solid-phase extraction device and interface requirements are provided.

Carbohydrates and Glycoproteins

Kasturi, L., Chen, H. and Shakin-Eshleman, S. H. (1997) Biochemical Journal 323, 415-419. Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors.

A long-recognized consensus sequence for N-linked glycosylation sites in proteins is NXS or NXT, where X is any amino acid residue except P. To more thoroughly define this consensus sequence, the effect of residue X on glycosylation was explored by site-directed mutagenesis. Interestingly, when X is W, D, E, or L, NXS is not frequently used as a glycosylation site, but these same residues make NXT an excellent site for carbohydrate attachment. This should prove useful when attempting to identify potential glycosylation sites in protein sequences.

DNA Sequence Analysis

Li, P., Kupfer, K. C., Davies, C. J., Burbee, D., Evans, G. A. and Garner, H. R. (1997) Genomics 40, 476-485. PRIMO: A Primer Design Program That Applies Base Quality Statistics for Automated Large-Scale DNA Sequencing.

Two computer programs are described for automated selection of primers for DNA sequencing projects. One program runs on a Unix platform and is intended for management of large-scale, genomic sequencing projects. The second program operates from a Macintosh platform: it automatically assigns "quality" values to analyzed data generated with ABI instruments to identify regions where primers for further sequencing can be made with confidence. Primers are then selected using a modification of the program Primer from the Whitehead Institute. Both programs are available from the authors.

Mass Spectrometry

Opiteck, G. J., Lewis, K. C. and Jorgenson, J. W. (1997) Analytical Chemistry 69, 1518-1524. Comprehensive On-Line LC/LC/MS of Proteins.

A test of the feasibility of using two-dimensional HPLC in place of two-dimensional electrophoresis for subsequent protein identification by mass analysis. In the first dimension, ten standard proteins were separated by cation-exchange HPLC. As they eluted, a two-position, eight-port valve directed them to a reversed-phase HPLC system, for a second dimension of separation, followed by downstream, on-line MS. Compared to the electrophoretic approach, the sample capacity and sensitivity of the HPLC system were limited, partly by a 10:1 flow split before mass analysis.

Cohen, S. L. and Chait, B. T. (1997) Analytical Biochemistry 247, 257-267. Mass Spectrometry of Whole Proteins Eluted from Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Gels.

A procedure for mass analyzing proteins up to 70 kDa from SDS-PAGE gels. After negative staining with copper or zinc, protein bands are excised and extracted with formic acid/water/isopropanol before analysis by MALDI-MS. With sample amounts greater than 25 pmol, the eluted sample is lyophilized and then resolubilized with the MALDI matrix solution. When the sample amount is less than 25 pmol, MALDI matrix is included in the eluting mixture and is allowed to slowly crystallize during the elution process. Using this method, 1 pmol (16 ng) of leptin could be analyzed.

van Oostveen, I., Ducret, A. and Aebersold, R. (1997) Analytical Biochemistry 247, 310-318. Colloidal Silver Staining of Electroblotted Proteins for High Sensitivity Peptide Mapping by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry.

A sensitive method for staining proteins on nitrocellulose or PVDF membranes. Staining is accomplished with colloidal silver, made from ferrous sulfate and silver nitrate in a sodium citrate buffer, and is capable of detecting as little as 5 ng of bound protein. The stained samples could then be digested on the membranes, without destaining, and mass analyzed for protein identification.

Peptides&emdash;Synthesis and Purification

Weber, P. J. A. and Beck-Sickinger, A. G. (1997) Journal of Peptide Research 49, 375-383. Comparison of the photochemical behavior of four different photoactivatable probes.

Photoaffinity labeling is a powerful method for mapping active sites. In addition, immunogens can be conveniently produced by coupling photoprobe-labeled peptides to carrier molecules, an approach made popular by the ability to incorporate photoactive compounds during solid-phase peptide synthesis. In this article, the properties of four photoactive probes are compared: irradiation conditions, cross-linking efficiency, likely byproducts, and the effects of water on stability and adduct formation. Overall, benzophenones and aryl diazirines provided the best incorporation with minimal side-reactions and by-products, compared to aryl azides and alpha-diazocarbonyls.

Roggero, M.A., Servis, C. and Corradin, G. (1997) FEBS Letters 408, 285-288. A simple and rapid procedure for the purification of synthetic polypeptides by a combination of affinity chromatography and methionyl chemistry.

A three-pronged strategy for purifying large, difficult peptides synthesized by Fmoc chemistry. The approach is to add a His6-Gly-Met tag to the amino-terminus of the peptide and replace internal methionyl residues with methionine sulfoxide. After cleaving the peptide from the synthesis resin, the full-length peptide is affinity purified, through its amino-terminal tag, on a Ni-chelate column. The tag is then removed by cleaving the eluted peptide with cyanogen bromide&emdash;cleavage will not occur at the methionine-sulfoxide residues, which are later reduced to methionine. This strategy was used to purify a 69-residue peptide.

Protein Characterization

Dunn, M. J. [editor] (1997) Electrophoresis 18, 305-662. From Protein Maps to Genomes. Proceedings of the Second Siena Two-Dimensional Electrophoresis Meeting.

An entire issue is devoted to the proceedings of this conference with full up-to-date state-of-the-art protein characterization techniques and analytical methods particularly relevant to proteome analysis.

Dong, M., Baggetto, L. G., Falson, P., LeMaire, M. and Penin, F. (1997) Analytical Biochemistry 247, 333-341. Complete Removal and Exchange of Sodium Dodecyl Sulfate Bound to Soluble and Membrane Proteins and Restoration of Their Activities, Using Ceramic Hydroxyapatite Chromatography.

A novel, efficient method to remove SDS from proteins for recovery of biological activity. SDS-protein complexes were bound to hydroxyapatite, and the SDS was "exchanged" by including mild detergents&emdash;such as dodecyl maltoside, CHAPS, or octaethyleneglycol dodecyl ether&emdash;in the low-phosphate buffer for washing the column. Proteins were then eluted by increasing the phosphate concentration. For two membrane proteins, 90% of activity was recovered, and for three soluble proteins, this value averaged about 97%.

Return to the The ABRF Home Page