On-line liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) using an LTQ-Orbitrap mass spectrometer was employed to investigate the metabolite profiles of a model siRNA duplex designated HBV263. The HBV263 duplex was incubated in rat and human serum and liver microsomes in vitro. The siRNA drug and its metabolites were then extracted using a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE), and analyzed by LC/ESI-MS. High-resolution accurate mass data enabled differentiation between two possible metabolite sequences with a monoisotopic molecular mass difference of less than 1 Da. ProMass deconvolution software was used to provide semi-automated data processing. In vitro serum and liver microsome incubation samples afforded different metabolite patterns: the antisense strand of the duplex was degraded preferentially in rat and human serum, while the sense strand of the duplex was less stable in rat and human liver microsomes.
Liquid chromatography/mass spectrometry (LC/MS) was used as a method for analyzing the metabolites of a model short interfering RNA (siRNA) duplex. The model siRNA duplex incorporated oligonucleotide stabilizing and protecting chemistries as these have been shown to increase the half-life of oligonucleotides. Two complementary 23 nucleotide single strands were joined to form the duplex. The intact duplex was analyzed using ion-pair reversed-phase chromatography coupled to electrospray ionization mass spectrometry (ESI-MS). The method used a hexafluoroisopropanol/triethylamine ion-pairing buffer with a methanol gradient to separate single-stranded oligonucleotide components from the duplex. This buffer system with ESI also preserved the duplex in the gas phase for analysis by a triple quadrupole mass spectrometer. Using this methodology, in vitro and in vivo metabolites from urine and rabbit ocular vitreous humor were determined and a pattern of duplex siRNA degradation was established. The masses of the metabolites were determined by ESI-MS and used with the known sequence of the siRNA duplex to identify the metabolites. Over the time course of the metabolism experiments it was shown that the breakdown products of the siRNA are consistent with the nuclease protection given by chemical modifications and that the duplex structure adds additional stability compared to the single strands alone. This study demonstrates that the ability of LC/MS to analyze duplex oligonucleotides has unique benefits for the study of siRNA metabolism.
Fatty acid methyl esters (FAMEs) were generated in situ, during pyrolysis, from whole-cell bacterial samples and analyzed by mass spectrometry (MS). The FAME profiles obtained by an in situ thermal hydrolysis methylation (THM) step were compared with gas chromatography (GC) and MS analyses of the chemically extracted and methylated fatty acids. This correlation was based on the ability of each technique to differentiate a representative group of 15 bacteria at the species level as predicted by principal component analysis. All three analyses, GC/FAME, pyrolysis-MS/FAME, and in situ THM-MS/FAME differentiated the studied bacterial sample set into three discrete clusters. The bacteria comprising each cluster were the same for all three analyses, showing that taxonomic information of the lipid profiles was preserved in the Py-MS/FAME and in situ THM-MS/FAME analyses of whole cells. Contributions from saturated, unsaturated, cyclopropyl, and branched bacterial fatty acids to the differentiation of microorganisms were identified for all three analyses. The in situ THM-MS/FAME approach is simple, requires small samples (approximately 2 x 10(6) cells/profile), and is rapid, with a total analysis time under 5 min/sample.
A label-free method for determining the 5'-end cap identity and orientation of a messenger RNA (mRNA) is described. Biotin-tagged probes that were complementary to the 5' end of target mRNA were used with RNase H to cleave the 5' end of the mRNA. The cleaved end sequence was isolated using streptavidin-coated magnetic beads and then analyzed by LC-MS. Quantitative and qualitative information on the 5' cap was determined from the unique mass of the isolated cleaved sequence. This approach, combined with the use of 5' RNA pyrophosphohydrolase, was also used to ascertain the orientation of the 5' cap. The assay showed low-picomole sensitivity for detecting capping reaction impurities. Uncapped triphosphate mRNA, spiked into 100 pmol of capped mRNA, could be detected over the tested range of 0.5 to 25 % with a linear response. The capping efficiency of several vaccinia-capped mRNA preparations was determined to be between 88 and 98 % depending on the modification type and length of the mRNA. mRNA of 2.2K and 9K nucleotides in length and containing the modified nucleotides pseudouridine and 5-methylcytidine were all successfully analyzed, demonstrating the utility of the technique to study mRNA capping. Graphical abstract mRNA 5' end analysis with RNAse H cleavage and capture probe.
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