A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from twodimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of twodimensional gel spots. The method was applied to a twodimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identiy.The identification of a purified protein is necessary in many areas of biochemical research. As the resolution and sensitivity of purification tools increase, the demand for protein sequencing increases. For example, a single high-resolution two-dimensional polyacrylamide gel can separate hundreds of proteins (1,2). Identification of all the resolvable proteins on a two-dimensional gel by conventional protein sequencing is a daunting task. The correlation of DNA from large-scale sequencing projects with their protein products will continue to place increasing demands upon protein sequencing.Proteins that are N-terminally blocked present an additional challenge since they cannot be directly sequenced by Edman degradation. Blockage may occur by posttranslational modification during protein synthesis or during purification. Many intracellular proteins have been reported to be N-terminally acetylated (3). In order to obtain internal sequence on a blocked protein, 50-100 pmol of material is usually required. The blocked protein is chemically or enzymatically cleaved. The peptides are then separated by HPLC and sequenced, a process which can take 3-4 days. In addition, proteins initially thought to be novel may, after purification and sequencing, be found already to exist in the protein sequence database. As a result, a significant fraction of sequencer time is spent simply identifying known proteins.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 1...

Retroviral genomes must dimerize to be fully infectious. Dimerization is directed by a unique RNA hairpin structure with a palindrome in its loop: hairpins of two strands first associate transiently through their loops, and then refold to a more stable, linear duplex. The structure of the initial, kissing-loop dimer from HIV-1, solved using 2D NMR, is bent and metastable, its interface being formed not only by standard basepairing between palindromes, but also by a distinctive pattern of interstrand stacking among bases at the stem-loop junctions. This creates mechanical distortions that partially melt both stems, which may facilitate spontaneous refolding of this RNA complex into linear form.

Various matrix mixtures have been used for matrix-assisted laser desorption/ionization mass spectrometry to characterize the tryptic maps of recombinant human growth hormone (rhGH) and recombinant human tissue plasminogen activator (rt-PA). Carbohydrate-containing comatrices give improved results over single-component matrices. Of those studied, fucose plus 2,5-dihydroxybenzoic acid (DHB) produced a signal for 24 out of 25, or 96%, of the tryptic peptides of rhGH in a single spectrum. These results were obtained for analyses of as little as 280 fmol of unfractionated material measured in digestion buffer. Analysis of 150 fmol showed a decrease in the relative abundance of higher molecular weight peptides. The incorporation of 5-methoxysalicylic acid (5MSA) as a comatrix in a molar ratio of analyte:fucose:DHB:5MSA = 1:5000:5000:50 gave signals for 45 out of 51 peptides for 4.5 pmol of a tryptic digest of rt-PA, corresponding to 88% of the expected fragments. Unobserved peptides were typically di- and tripeptides. Three glycopeptides were observed with peaks corresponding to the known major glycoforms. The fucose/DHB and 5MSA/DHB comatrices produced significant enhancements in spectral quality over DHB alone, including suppression of matrix peaks, increased ion signal, improved resolution, increased number of useful laser shots per crystal, and minimization of baseline slope. Spectra obtained with fucose/DHB generally surpassed DHB/5MSA in quality, though both matrix mixtures were clearly superior to neat DHB. Fucose/DHB demonstrated an increase in tolerance to ionic contaminants by producing a 10-fold reduction in the abundance of (M + Na)+ions. A trimatrix, DHB/5MSA/fucose, produced the highest quality spectra to date, although only marginally better than the fucose/DHB comatrix.

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