Sequence Information from 42−108-mer DNAs (Complete for a 50-mer) by Tandem Mass Spectrometry

Modified oligonucleotides continue to play an important role as antisense compounds that inhibit the expression of genes associated with metabolic disorders, cancer, and infectious diseases. Because the majority of modifications render these molecules refractory to standard enzymatic sequencing techniques, alternative sequencing methods which are fast and reliable are needed. In this work we explore how sugar and backbone modifications affect fragmentation patterns observed from oligonucleotides which are fragmented by infrared multiple photon dissociation in the external reservoir of an electrospray ionization Fourier transform ion cyclotron mass spectrometer. The modifications influence which fragment types (i.e., a n -B versus c n ) dominate and the ease with which the oligonucleotides are fragmented. General observations for confirming the sequence of oligonucleotides are described. A ntisense compounds are modified oligonucleotides that bind messenger RNA and inhibit the expression of genes associated with metabolic disorders, cancer, and infectious diseases. Modified oligonucleotides are necessary to increase the binding of the oligonucleotides to the messenger RNA as well as to minimize the in vivo enzymatic degradation of the oligonucleotides [1]. Typically, modifications are made to the phosphate backbone and to the 2Ј position of the sugar. Fast and reliable methods are needed for sequencing these oligonucleotides since standard enzymatic techniques are not compatible with the majority of these modifications.Mass spectrometry has developed into a powerful tool for studying macromolecules of biological importance, including nucleic acids. Soft ionization techniques such as electrospray ionization [2-4] and matrixassisted laser desorption/ionization [5,6] allow the introduction of large intact biomolecules into the gas phase for mass spectrometric analysis. In addition to accurate molecular weight determination, mass spectrometry in combination with different fragmentation schemes provides information about sequence and/or structure of the biomolecules. Seminal work by McLafferty and coworkers demonstrated the ability to obtain partial sequence information for DNA oligonucleotides up to 108 nucleotides in length using tandem mass spectrometry techniques on a FTICR spectrometer [7]. By combining various fragmentation schemes, it was shown that complete sequence coverage could be obtained for a 50-mer DNA [7][8][9]. Other researchers demonstrated that sequence information and identification of modified bases could be obtained by combining fragmentation schemes with ion trap mass spectrometry [10] and triple quadrupole mass spectrometry [11][12][13].Based on the small amounts of sample required and the unambiguous signatures generated, mass spectrometry is emerging as the preferred platform on which to interrogate and sequence modified oligonucleotides. In order to faithfully sequence oligonucleotides containing desperate chemical modifications, an understanding of how the modifications influence the fragme...

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confidence: 99%

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confidence: 99%

Sequence confirmation of modified oligonucleotides using IRMPD in the external ion reservoir of an electrospray ionization fourier transform ion cyclotron mass spectrometer

Sannes‐Lowery

Hofstadler

2003

“…Additional advances included external ion accumulation [11], insertion of an external quadrupole for mass selection prior to ion accumulation and detection [12][13][14], and application of a dc voltage gradient to facilitate ion transfer to the ICR cell [12,15]. Dissociation techniques such as collision-assisted dissociation (CAD) [16], infrared multiphoton dissociation (IRMPD) [17], and electron capture dissociation (ECD) [18] can provide extensive backbone bond cleavage for proteins [19] and DNA oligomers [20] for highly efficient analysis of biomolecules in a "topdown" fashion [21,22].…”

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confidence: 99%

Construction of a hybrid quadrupole/fourier transform ion cyclotron resonance mass spectrometer for versatile MS/MS above 10 kDa

Patrie

Charlebois

Whipple

et al. 2004

107

Technological advancements including an open-cylindrical Penning trap with capacitively coupled ICR cell, selective ion accumulation with a resolving quadrupole, and a voltage gradient used during ion extraction from an octopole ion trap, have individually improved dynamic range and sensitivity in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). Documented here is a new instrument utilizing these technologies toward the robust detection and fragmentation of biomolecules Ͼ10 kDa. Up to 55-fold enhancement in ion population by selective ion accumulation combined with 10-to 20-fold signal-to-noise improvement by application of a DC voltage gradient to an accumulation octopole during the ion transfer event offers improved signal-to-noise (or speed) of MS/MS experiments, for proteins from Methanococcus jannaschii and Saccharomyces cerevisiae whole cell lysates. After external quadrupole filtering with a 40 m/z window, three proteins were fragmented (and identified) in parallel from the database of Methanococcus jannaschii. Electron capture dissociation (ECD) of an intact yeast protein provides extensive sequence information resulting in a high degree of localization for an N-terminal acetylation. Hybrid fragmentation, infrared multiphoton dissociation (IRMPD) followed by low energy electrons (ECD), with the electron source located laterally off the z-axis and external to the magnet bore, presents a strategy for identification of proteins by means of the sequence tag approach. Automated implementation of diverse MS n approaches in a Q-FTMS instrument promises to help realize "top-down" proteomics in the future. (J Am Soc Mass Spectrom 2004, 15, 1099 -1108

“…Elucidating these mechanisms is of practical importance, since it will facilitate the rational development of MS-based techniques for sequencing. In addition, the dissociation of biomolecules in the gas phase reflects their intrinsic properties, which are of fundamental interest.For oligodeoxynucleotides (ODNs), sequence information can be obtained from the fragmentation behaviour of either the protonated or deprotonated gaseous ions and the dissociation behaviour of both forms has been extensively investigated [1][2][3][4][5][6][7][8][9][10]. The dissociation of deprotonated ODNs, the focus of the present study, has been shown to proceed first by the loss of a nucleobase, adenine (A), guanine (G), cytosine (C) or thymine (T), in its neutral or deprotonated form, followed by fragmentation of the phosphoester bond at the deoxyribose 3Ј C™O bond at the site of base loss to produce (a-base) and w type ions, according to the nomenclature proposed by McLuckey and coworkers [7].…”

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confidence: 99%

“…However, in this same study the authors noted different trends at higher charge states: A Ͼ C Ϸ G ӷ T (-4); A Ͼ C Ͼ G ӷ T (-5). McLafferty and coworkers, using infrared multiphoton dissociation (IRMPD) and CID implemented with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR/MS), observed the following trend for large multiply deprotonated oligonucleotides (up to 100 mers): A Ͼ C Ϸ G ӷ T [9]. In contrast, McLuckey et al did not observe any preferential loss of the nucleobases in CID-ITMS experiments perform on small, but relatively highly charged, ODN anions (4-, 5-and 8-mers) [8].…”

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confidence: 99%

Arrhenius activation parameters for the loss of neutral nucleobases from deprotonated oligonucleotide anions in the gas phase

Daneshfar

Klassen

2004

Arrhenius activation parameters (E a and A) for the loss of neutral nucleobase from a series of doubly deprotonated oligodexoynucleotide 10-mers of the type XT 9 , T 9 X, and T 5 XT 4 , where X ϭ A, C, and G, have been determined using the blackbody infrared radiative dissociation technique. At temperatures of 120 to 190°C, the anions dissociate exclusively by the loss of a neutral nucleobase (XH), followed by cleavage of the sugar 3Ј C™O bond leading to (a-XH) and w type ions or, in the case of the T 9 X 2Ϫ ions, the loss of H 2 O. The dissociation kinetics and energetics are sensitive to the nature and position of X. Over the temperature range investigated, the kinetics for the loss of AH and GH were similar, but ϳ100 times faster than for the loss of CH. For the loss of AH and GH, the values of E a are sensitive to the position of the base. The order of the E a s for the loss of XH from the 5Ј and 3Ј termini is: C Ͼ G Ͼ A; while for T 5 XT 4 the order is: C Ͼ A Ͼ G. The trends in the values of E a do not parallel the trend in deprotonation enthalpies or proton affinities of the nucleobases in the gas phase, indicating that the energetic differences do not simply reflect differences in their gas phase acidity or basicity. The pre-exponential factors (A) vary from 10 10 to 10 15 s Ϫ1 , depending on the nature and position of X. These results suggest that the reactivity of individual nucleobases is influenced by stabilizing intramolecular interactions. M ass spectrometry (MS), combined with soft ionization techniques such as electrospray (ES) and matrix assisted laser desorption/ ionization (MALDI), has become an indispensable tool for identifying the primary structure (sequence) of biopolymers: peptides, oligosaccharides, and oligonucleotides. The mass spectrometry-based sequencing approach typically involves isolating the biopolymer ion of interest in the gas phase, dissociating it to produce sequence specific fragment ions and accurately determining the mass of the ions. Sequence information is extracted from the mass differences of the sequential fragment ions of the same general structure. The MSbased sequencing approach has many attractive features, most notably its inherent speed and sensitivity and its ability to sequence biopolymers containing unnatural or unusual modifications. Despite its widespread use in the sequencing of biopolymers, fundamental questions regarding the gas phase dissociation mechanisms remain. Elucidating these mechanisms is of practical importance, since it will facilitate the rational development of MS-based techniques for sequencing. In addition, the dissociation of biomolecules in the gas phase reflects their intrinsic properties, which are of fundamental interest.For oligodeoxynucleotides (ODNs), sequence information can be obtained from the fragmentation behaviour of either the protonated or deprotonated gaseous ions and the dissociation behaviour of both forms has been extensively investigated [1][2][3][4][5][6][7][8][9][10]. The dissociation of deprotonated ODNs, the focus ...