The 5' noncoding region of poliovirus RNA contains an internal ribosome entry site (IRES) for capindependent initiation of translation. Utilization of the IRES requires the participation of one or more cellular proteins that mediate events in the translation initiation reaction, but whose biochemical roles have not been defined. In this report, we identify a cellular RNA binding protein isolated from the ribosomal salt wash of uninfected HeLa cells that specifically binds to stem-loop IV, a domain located in the central part of the poliovirus IRES. The protein was isolated by specific RNA affinity chromatography, and 55% of its sequence was determined by automated liquid chromatography-tandem mass spectrometry. The sequence obtained matched that of poly(rC) binding protein 2 (PCBP2), previously identified as an RNA binding protein from human cells. PCBP2, as well as a related protein, PCBP1, was over-expressed in Escherichia coli after cloning the cDNAs into an expression plasmid to produce a histidine-tagged fusion protein. Specific interaction between recombinant PCBP2 and poliovirus stem-loop IV was demonstrated by RNA mobility shift analysis. The closely related PCBP1 showed no stable interaction with the RNA. Stem-loop IV RNA containing a three nucleotide insertion that abrogates translation activity and virus viability was unable to bind PCBP2.

A microcapillary liquid chromatography (HPLC) system designed for the gradient elution of peptide and protein samples at flow rates < 1 microL/min has been coupled to a triple-sector quadrupole mass spectrometer via a simple sheathless electrospray interface (microspray). The microspray interface used a flame-drawn, uncoated, fused silica needle with tip outer diameters in the range of 15-20 microm and an opening less than 5 microm in diameter. Online sample filtration to prevent clogging of the drawn needle was accomplished by using a hydrophilic PVDF membrane filter integrated into the needle assembly. The spray potential (0.5-1 kV) was applied directly to the sample stream through the capillary union. Stable electrospray conditions were obtained over the full range of the gradient (0-90% acetonitrile in water) and was generally independent of flow rate. Both off-line and online analyses of proteins and peptide digest mixtures were performed at sample levels less than 10 fmol. HPLC parameters could be optimized for either rapid LC/MS analysis or enhanced performance in LC/MS/MS experiments by modulation of the eluting peak widths. Additionally, flow could be greatly reduced as selected components pass through the interface to prolong the time available to collect mass spectral data. The reduced spectral background and peak width manipulation facilitated the acquisition of peptide production spectra (MS/MS) using real-time, automated instrument control procedures.

The structural characterization of proteins and peptides isolated in minute quantities requires the most efficient use of available sample. A mass spectrometer data system was programmed to continuously evaluate incoming liquid chromatography/mass spectrometry data against a user-defined array of information. The resulting conclusions were used to automatically set and modify acquisition parameters in real time to collect collision-induced dissociation spectra for selected ions (tandem mass spectrometry). This approach has provided a mechanism to target specific subsets of masses in a complex mixture and/or to discriminate selectively against masses that are known or not of interest. Masses of contaminants or peptide masses derived from known proteins can be automatically recorded and removed from further consideration for collision-induced dissociation analysis. Once recorded, these "libraries" of masses can be used across multiple analyses. This technique directs the mass spectrometer data system to focus on the analysis of masses significant to the user, even if their signal intensities are well below the intensities of contaminating masses. When combined with a database search program to correlate tandem mass spectra to known protein sequences, the identity of the protein can be established unequivocally by using less than 100 fmol of sample.

On-line capillary liquid chromatography-electrospray mass spectrometry and tandem mass spectrometry, in combination with precursor feeding and acetylation studies, were used to identify and characterize the siderophores of the fire blight pathogen Erwinia amylovoru. Proferrioxamines D, , El G,, G , , X, and X, were found, with D, and E being the major siderophores for all strains of E. amylovoru studied. These proferrioxamines have previously been observed with other microbial species. In addition, eleven additional hydroxamate compounds were found, nine of which occur naturally and two which were induced by directed fermentation. The novel natural proferrioxamines included three cyclic tetrahydroxamates, designated T,-,. The most abundant of these, T,, consists of four 5succinylamino-1-hydroxyaminopentane residues. In T, and T,, one of these residues is substituted by 4 succinylamino-1-hydroxyaminobutane and 3-succinylamino-l-hydroxyaminopropane, respectively. E. amylovora also incorporated Isuccinylamino-1-hydroxyaminopropane into a novel trihydroxamate, which has been designated X, . The novel finding that proferrioxamines may partly be comprised of 1,fdiaminopropane residues is noteworthy because previous feeding studies with Streptomyces olivaceus Tii 2718 suggested that 1,fdiaminopropane is biosynthetically not tolerated. A truncated proferrioxamine G , was also identified, which is referred to as G,,, and, on feeding of diaminobutane, tetrahydroxamates T, and T, , which are derived from T, by substitution of two and three diaminopentane with diaminobutane residues. The structures of five other hydroxamates, with molecular masses of 5-30 (X,), 544 (X9), 622 (T.,), 719 (T,) and 733 (T6), are not yet known. The results prove that the biosynthetic capabilities of E. amylovoru for proferrioxamine production are flexible. E. amylovora may therefore be a good organism for the fermentative production of novel proferrioxamines that may be useful for reversing or avoiding aluminum and iron intoxications in man, for tumor diagnosis and therapy with radiolabeled antibodies and for use as DNA-cleaving reagents. On the other hand, the production of all the different proferrioxamines involves common biosynthetic steps, some of which are not used in higher plants or vertebrates. Interference with the biosynthesis of proferrioxamines may therefore provide an opportunity for the development of alternative fire blight control agents.