Top-down mass spectrometry for sequencing of larger (up to 61 nt) RNA by CAD and EDD

Abstract

We have studied the effect of solution additives on hydrolysis and charge state distribution in ESI MS of RNA. Lower and higher charge state ions can be electrosprayed from solutions containing 25 mM piperidine/25 mM imidazole and 1% vol. triethylamine, respectively, with base-catalyzed hydrolysis rates that are sufficiently slow to perform MS/MS experiments. These lower and higher charge state ions are suitable as precursors for CAD and EDD, respectively. We demonstrate nearly complete sequence coverage for 61 nt RNA dissociated by CAD, and 34 nt RNA dissociated by EDD, and suggest a mechanism for backbone fragmentation in EDD of RNA. (J Am Soc Mass Spectrom 2010, 21, 918 -929) © 2010 American Society for Mass Spectrometry T he "top-down" approach is increasingly being applied in mass spectrometry (MS) studies of proteins [1][2][3][4][5], using mostly collisionally activated dissociation (CAD) [6 -8], electron capture dissociation (ECD) [9 -12], or electron-transfer dissociation (ETD) [13] for generation of sequence-informative fragment ions from backbone cleavage in multiply protonated precursor ions. First examples of top-down mass spectrometry of smaller (up to 25 nt) multiply deprotonated desoxyribonucleic acids (DNA) were reported in the early 1990s [14 -16], and the concept was soon extended to larger (up to 108 nt) DNA [17].For ribonucleic acids (RNAs), the development and application of mass spectrometry-based methodology has been much slower than for DNA [18], possibly because the significance of RNA in regulation of gene expression was recognized only recently [19]. Topdown MS has been applied to study RNA modified by structural probes [20], for characterization of conserved domains of the HIV-1 packaging signal RNA [21], for investigating aptamer/ligand complexes [22], and binding of antibiotics to ribosomal RNA subdomains [23]. However, sequencing by top-down mass spectrometry of RNA Ͼ 20 nt has been demonstrated only recently, as discussed below.Collisionally activated dissociation of multiply deprotonated RNA from electrospray ionization (ESI) [24] has lately provided full sequence coverage for small interfering RNA (siRNA) [25] and a riboswitch aptamer domain sequence [26] consisting of 21 and 34 nucleotides (nt), respectively. Mass spectral quality, particularly with regard to undesired secondary fragmentation, was shown to critically depend on precursor ion charge [25,27] and collisional cooling of primary fragments [26]. Our rationale for the decrease in secondary fragmentation was that reduced net charge and collisional cooling minimize the internal energy of fragment ions from primary backbone cleavage, which makes them less prone to secondary fragmentation [26]. In our study of 34 nt RNA, we used ions of relatively low net charge, (M Ϫ 7H) 7Ϫ , which were electrosprayed from acidified solutions [26].However, mass spectrometer performance generally decreases with increasing mass-to-charge ratio, i.e., lower ion charge. An alternative method that was reported to actually give higher yields...

Bioengineering Novel Chimeric microRNA-34a for Prodrug Cancer Therapy: High-Yield Expression and Purification, and Structural and Functional Characterization

“…The procedures described by Taucher and Breuker (2010) were followed. The instrumental settings were optimized by automatic tuning with poly-d(T) 80 .…”

Chimeric MicroRNA-1291 Biosynthesized Efficiently in Escherichia coli Is Effective to Reduce Target Gene Expression in Human Carcinoma Cells and Improve Chemosensitivity

“…The procedure described previously (Taucher and Breuker, 2010) was followed for electrospray ionization (ESI)-MS analysis of intact ncRNAs. In particular, the electrospray solution consisted of 1 mM ncRNA, 25 mM imidazole, and 25 mM piperidine in 1:1 water/methanol.…”

Mass spectrometry in the biology of RNA and its modifications

“…Lastly, electron detachment dissociation [61,62] and infrared multiphoton dissociation [63] studies suggest that these fragmentations techniques may give valuable sequence information complementary to CID-obtained ditto, but these techniques were so far restricted to studies of model compounds.…”