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...

In recent years, new classes of nonprotein-coding ribonucleic acids (ncRNAs) with important cellular functions have been discovered. Of particular interest for biomolecular research and pharmaceutical developments are small ncRNAs that are involved in gene regulation, such as small interfering RNAs (21-28 nt), pre-microRNAs (70-80 nt), or riboswitches (34-200 nt). De novo sequencing of RNA by top-down mass spectrometry has so far been limited to RNA consisting of up to approximately 20 nt. We report here complete sequence coverage for 34 nt RNA (10.9 kDa), along with 30 out of 32 possible complementary ion pairs from collisionally activated dissociation (CAD) experiments. The key to minimizing undesired base loss and internal fragmentation is to minimize the internal energy of fragment ions from primary backbone cleavage. This can be achieved by collisional cooling of primary fragment ions and selection of precursor ions of relatively low negative net charge (about -0.2/nt).

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Electron detachment dissociation (EDD) is an emerging mass spectrometry (MS) technique for the primary structure analysis of peptides, carbohydrates, and oligonucleotides. Herein, we explore the potential of EDD for sequencing of proteins of up to 147 amino acid residues by using top-down MS. Sequence coverage ranged from 72 % for Melittin, which lacks carboxylic acid functionalities, to 19 % for an acidic 147-residue protein, to 12 % for Ferredoxin, which showed unusual backbone fragmentation next to cysteine residues. A limiting factor for protein sequencing by EDD is the facile loss of small molecules from amino acid side chains, in particular CO2. Based on the types of fragments observed and fragmentation patterns found, we propose detailed mechanisms for protein backbone cleavage and side chain dissociation in EDD. The insights from this study should further the development of EDD for top-down MS of acidic proteins.

All rights reserved EuroPEAN JourNAL of MASS SPEctroMEtry the majority of mass spectrometry-based approaches utilizes positively charged ions. for peptide or protein identification, the most commonly used method is collisionally activated dissociation (cAD) 1-4 of their (M + nH) n+ ions, typically formed by electrospray ionization (ESI). 5,6 More extensive protein sequence information can be obtained by top-down mass spectrometry 7-12 using electron capture dissociation (EcD), [13][14][15][16] including the identification and localization of post-translational modifications (PtMs). [17][18][19] EcD generally requires precursor ions carrying multiple positive net charges. However, many proteins have isoelectric points (pI) between ~4 and ~6 (figure 1 Acidic proteins and nucleic acids such as RNA are most readily ionized in electrospray ionization (ESI) operated in negative-ion mode.The multiply deprotonated protein or RNA ions can be used as precursors in top-down mass spectrometry. Because the performance of the dissociation method used critically depends on precursor ion negative net charge, it is important that the extent of charging in ESI can be manipulated efficiently. We show here that (M − nH) n− ion net charge of proteins and RNA can be controlled efficiently by the addition of organic bases to the electrosprayed solution. Our study also highlights the fact that ion formation in ESI in negative mode is only poorly understood.