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Changes in the mutual orientation of tRNA and 23S rRNA at the peptidyl transferase center of the ribosome detected by crosslinking of a photoreactive transition-state analog
Dynamic interactions between the amino acid acceptor end of tRNA and the ribosome underlie the synthesis of successive peptide bonds at the peptidyl transferase center (PTC) of the 50S ribosomal subunit. Photo-crosslinking of the 3′-terminal nucleotide of tRNA, which is adjacent to the attached amino acid or peptide, to components of the 50S subunit has proven to be a sensitive means for identifying specific protein and RNA segments in close proximity to the site of peptide bond formation. I have used this approach to follow changes in the position of the tRNA during peptide bond formation using several photoreactive tRNA-derived ligands. Three new photoreactive tRNA derivatives have been synthesized for use as probes of the PTC of the ribosome. In two of these derivatives, the 3′ adenosine in position 76 of yeast tRNAPhe has been replaced by either 2-azidodeoxyadenosine or 2-azido-2′-O-methyladenosine, while in a third the 3′-terminal 2-azidodeoxyadenosine of the tRNA is joined to puromycin via a phosphoramidate linkage to generate a photoreactive transition-state analog. All three derivatives bind to the P site of 70S ribosomes with affinities similar to that of unmodified tRNA Phe and can be crosslinked to components of the 50S ribosomal subunit by irradiation with near UV light. Yeast tRNAPhe containing 2-azidoadenosine, [2N3A76]tRNAPhe, typically crosslinks to the N-terminal sequence of protein L27 as well as to nucleotides U2506 and U2585 of the 23S rRNA. While the photoreactive transition-state analog, [2N3dA76]tRNAPhe-p-Puro, crosslinked the same components as [2N3A76]tRNAPhe, the distribution of crosslinks is altered significantly. The crosslinking to nucleotide U2506 is strongly reduced, and two new crosslinked nucleotides A2450 and A2602 were detected. Characteristic differences in the crosslinking patterns suggest that these tRNA derivatives can be used to follow subtle changes in the position of the tRNA relative to the components of the PTC
Comparability analysis of protein therapeutics by bottom-up LC-MS with stable isotope-tagged reference standards
Comparability studies lie at the heart of assessments that evaluate differences amongst manufacturing processes and stability studies of protein therapeutics. Low resolution chromatographic and electrophoretic methods facilitate quantitation, but do not always yield detailed insight into the effect of the manufacturing change or environmental stress. Conversely, mass spectrometry (MS) can provide high resolution information on the molecule, but conventional methods are not very quantitative. This gap can be reconciled by use of a stable isotope-tagged reference standard (SITRS), a version of the analyte protein that is uniformly labeled with 13C6-arginine and 13C6-lysine. The SITRS serves as an internal control that is trypsin-digested and analyzed by liquid chromatography (LC)-MS with the analyte sample. The ratio of the ion intensities of each unlabeled and labeled peptide pair is then compared to that of other sample(s). A comparison of these ratios provides a readily accessible way to spot even minute differences among samples. In a study of a monoclonal antibody (mAb) spiked with varying amounts of the same antibody bearing point mutations, peptides containing the mutations were readily identified and quantified at concentrations as low as 2% relative to unmodified peptides. The method was robust, reproducible and produced a linear response for every peptide that was monitored. The method was also successfully used to distinguish between two batches of a mAb that were produced in two different cell lines while two batches produced from the same cell line were found to be highly comparable. Finally, the use of the SITRS method in the comparison of two stressed mAb samples enabled the identification of sites susceptible to deamidation and oxidation, as well as their quantitation. The experimental results indicate that use of a SITRS in a peptide mapping experiment with MS detection enables sensitive and quantitative comparability studies of proteins at high resolution