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Amino Acid and Sequence Analysis

Gooley AA, Ou K, Russell J, Wilkins MR, Sanchez JC, Hochstrasser DF, Williams KL. A role for Edman degradation in proteome studies. Electrophoresis 1997;18: 1068-72.

Further work from this group on basic technologies for proteome analysis. Previously, the authors have described how short, 3- to 5-residue Edman sequence tags can be used to identify unknown proteins from two-dimensional gels. Here, the authors describe two modifications that can be made to common, widely used commercial instruments to increase automated sample throughput. First, membrane-bound samples are fed to the instrument with a 16-stage carousel. Second, each reaction cartridge can be loaded with as many as four different unknown proteins; as long as the proteins have widely different molecular weights and are present at similar intensities on the gel, they are often distinguishable by their initial yields.

Mo B, Li J, Liang S. Chemical carboxy-terminal sequence analysis of peptides using acetyl isothiocyanate. Analytical Biochemistry 1997;252:169-76.

This paper describes an easily made, useful reagent for carboxyl-terminal sequencing, acetyl isothiocyanate. This reagent both activates and derivatizes the carboxyl terminus, without an oxazolinone intermediate, and gives more complete reactions than similar reagents. Including organic alkaline catalysts in the reaction mix improves this reagent's reactivity.

Mass Spectrometry

Jones MD, Patterson SD, Hsieng SL. Determination of disulfide bonds in highly bridged disulfide-linked peptides by matrix-assisted laser desorption/ionization mass spectrometry with postsource decay. Analytical Chemistry 1998;70:136-43.

The difficulty in assigning disulfide bonds in multichain peptide complexes with the structure A-B-C is often focused on peptide B: which of its Cys residues is linked to peptide A and which to peptide C? Traditionally, these assignments are made by Edman sequencing, after multiple rounds of proteolysis and HPLC, consuming large sample amounts. This report shows that under certain conditions reflectron MALDI mass analysis can reveal precisely how these peptide complexes are linked together, through sequence-dependent post-source decay patterns. The key to this is fragmentation at peptide B's polypeptide backbone between the two bridged Cys residues during post-source decay, to produce ions consistent with only one possible structure.

Qin J, Fenyo D, Zhao Y, Hall WW, Chao DM, Wilson CJ, Young RA, Chait BT. A strategy for rapid, high-confidence protein identification. Analytical Chemistry 1997;69:3995-4001.

Previously these authors have reported that tryptic peptides tend to fragment at Asp and Glu residues in a MALDI-ion trap mass spectrometer during tandem mass analysis. Here they show this property is not a limitation but is useful for identifying unknown proteins in gels, because it contains more specific information than peptide mass alone and less complex information than a complete fragmentation spectrum.

Qin J, Chait BT. Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry. Analytical Chemistry 1997;69:4002-9.

Application of the MALDI-ion trap spectrometer to peptides containing native post-translational modifications or non-native chemical modifications. Single-stage analysis with a MALDI-ion trap instrument appears to show these modifications more readily than other mass analysis methods do: because the measurement time-scale is relatively long, the spectrum shows strong signals for fragmentation at low-energy bonds, such as protein modification sites. Examples are shown for disulfide bonds in multichain peptides, phosphorylation, glycosylation, methionine oxidation, and photo-crosslinking.

Peptides-Synthesis and Purification

Severin K, Lee D, Martinez J, Ghadiri M. Peptide self-replication via template-directed ligation. Chem. Eur. J. 1997;3:1017-24.

This paper describes an intriguing model in which peptides, rather than nucleic acids, show certain limited self-replicating behavior. The template peptide has a heptad pseudo-repeat structure, (abcdefg)_n, in which residues a and d are hydrophobic. Such peptides interact hydrophobically to form coiled coil dimers. A pair of shorter peptides, with sequences identical to the N- and C-terminal halves of the template, are allowed to anneal to the template peptide. The N-terminal half has a strongly electrophilic group at its C-terminus, and the C-terminal half has a strongly nucleophilic group at its N-terminus. Upon annealing to the template, these groups are in close proximity and react to form a peptide bond, an application of the chemistry originally developed by Steve Kent. The resulting peptide can become the template for the synthesis of a new strand, and so on, in autocatalytic fashion.

Yao S, Ghosh I, Zutshi R, Chmielewski J. A pH-modulated, self-replicating peptide. J. Am. Chem. Soc. 1997;119: 10559-60.

In work similar to that described above, glutamic acid residues are placed at positions e and g in the heptad repeat. At neutral pH values, the negative charges on the glutamic acid side chains destabilize the coiled coil structure, whereas, at acidic pH, these side chains are protonated and the stability of the coiled coil is restored. As expected, autocatalytic, template-directed ligation occurs at acidic pH, but is suppressed as the pH is raised.

Crimmins DL. Strong cation-exchange high-performance liquid chromatography as a versatile tool for the characterization and purification of peptides. Analytica Chimica Acta 1997;352:21-30.

A review of strong cation exchange HPLC methods for peptide separation, with examples to show their usefulness as a complement to reverse phase HPLC. Typical conditions employ a commercially available sulphoethyl aspartamide stationary phase, acidic phosphate buffers, and salt-gradient elution. N-terminally blocked peptides are shown to be readily separated from the corresponding unblocked peptides, and disulphide linked peptides from the corresponding unlinked ones. The polarity of uncharged chemical groups as well as differences in numbers of positive charges is shown to contribute to separations. Good resolution of a protein digest made with endoproteinese Glu-C is illustrated.

Lew S, London E. Simple procedure for reversed-phase high-performance liquid chromatographic purification of long hydrophobic peptides that form transmembrane helices. Analytical Biochemistry 1997;251:113-16.

Here is an approach to making reverse-phase analysis of very hydrophobic peptides less frustrating. First, determine the minimum concentration of isopropanol needed to dissolve the peptide in the presence of 0.1 % TFA. Apply this solution to a column equilibrated with a mobile phase of the same composition so that the peptide remains in solution. Then elute it by increasing the isopropanol concetration very slowly (0.2-0.8 % per minute).

Protein Characterization

Haeberle JR. High-temperature sodium dodecyl sulphate polyacrylamide gel electrophoresis. BioTechniques 1997;23:638-40.

This article suggests performing SDS-polyacrylamide gel electrophoresis at high temperature - on purpose! The electrophoretic mobility of ions depends strongly on temperature. This is why the uneven heating that occurs when gels are run at too high a current causes "smiling". However, if the gel is heated evenly, electrophoresis can be performed much more rapidly with little penalty in resolution. This paper shows that electrophoresis times can be reduced from 2.5 hr. at 15°C to 20 min. at 70°C.

Liang FT, Granstrom DE, Timoney JF, Shi YF. Micropreparative high resolution purification of proteins by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and membrane blotting. Analytical Biochemistry 1997;250:61-65.

O'Farrell two-dimensional gels provide superb resolution of complex mixtures of proteins, but are difficult to scale up for preparative purposes. This paper shows that isoelectric focusing can be used in conjunction with SDS-electrophoresis in the reverse of the normal order if, after performing SDS-electrophoresis, the proteins are electroblotted to a membrane of PVDF or nitrocellulose and washed prior to the isoelectric focusing step. The resulting two-dimensional separation is of high quality and can readily be scaled up.

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