Sodium controlled selective reactivity of protonated deoxy-oligonucleotides in the gas phase

Helga Dgg Flosadttir 0 1 Michal Stano 0 1 2 Oddur Inglfsson 0 1 0 Address reprint requests to Dr. O. Inglfsson, Department of Chemistry, University of Iceland, Science Institute , Dunhaga 3, Reykjavk 107 Iceland 1 Department of Chemistry, University of Iceland, Science Institute , Reykjavk, Iceland 2 Permanent address: Faculty of Mathematics, Physics , and Informatics, Department of Experimental Physics, Comenius University Mlynska Dolina F2 , Bratislava 84248, Slovakia Metastable fragmentation of the positively charged, hexameric oligonucleotides 5=-d(TTXYTT) (X and Y are dC, dG, or dA) and 5=-d(CTCGTT), 5=-d(TTCGTC) and 5=-d(CTCGTC) is studied after matrix assisted laser desorption/ionization (MALDI). The influence of the degree of sodiation, i.e., when the acidic protons are one by one exchanged against sodium ions, is systematically studied for the exchange of up to seven protons against sodium ions. Exchanging the acidic protons against sodium gradually quenches the backbone cleavage through the w and a-B channels, and quantitative quenching of these channels is generally achieved with the exchange of four protons against sodium ions. At the same time, the exchange of protons against sodium ions promotes the loss of a neutral, high proton affinity base. The formation of the w and a-B fragments is found to be highly dependent on the sequence of the central bases. A single mechanism consistent with these observations is proposed. In addition to the quenching of the classical w and a-B reaction channels, a drastic and abrupt on/off-switching of new reaction channels is observed as the degree of sodiation successively increases. These channels involve selective loss of the two central bases and the excision of a phosphodiester group and a sugar unit from the center of the oligonucleotides. Synchronously, the two terminal fragments recombine to form a tetramer containing the two terminal nucleosides from each end of the hexamer. Possible mechanism explaining these remarkable channels are discussed. (J Am Soc Mass Spectrom 2009, 20, 689 - 696) 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry - Tspectrometric techniques to shed light on the he need for new analytical methods and mass complex molecular structure and composition of biologically relevant molecules has drawn the attention of physicists and physical chemists more in the direction of gas-phase studies on molecules such as DNA, RNA, proteins, and peptides. The introduction of the electrospray ionization (ESI) and the matrix-assisted laser desorption ionization (MALDI) in the early 90s and the further development of these methods throughout the last decade have revolutionized the studies of biologically relevant molecules. Especially in the field of proteomics, these methods have proven to be very powerful both for the identification of known proteins and for the sequencing of unknown proteins and peptides. The metastable decay of preselected parent ions in MALDI has also proven to be very useful in proteomics [13], and is today a key technique in sequence analysis in proteomics. However, at the same time, the performance of these methods has not met expectations when it comes to the analyses of DNA segments. One of the difficulties hampering the use of MALDI TOF-MS in DNA analyses is the metastable and prompt decay of these molecules in the MALDI process [4 11]. Furthermore, the strong affinity of oligonucleotides to exchange their numerous acidic protons against sodium complicates the sample preparation considerably and often sets limits to the achievable resolution [4, 5]. In the last decades, several studies have been conducted to attempt to unravel the mechanism behind the prompt and metastable decay of oligonucleotides in MALDI [57, 9 26]. The bulk of this work is reviewed by Wan and Gross in 2001 [13] and by Wu and McLuckey in 2004 [6]. Several different mechanisms have been suggested for the decay of negatively charged oligonucleotides studied, and the experimental data does not appear to support one single mechanism [9, 10, 1215]. For the protonated oligonucleotides, on the other hand, the bulk of the studies conducted show that the fragmentation (with the exception of the loss of terminal nucleotides or nucleosides and the loss of water) is initiated by the protonation of a high proton affinity base [6, 11, 14, 24 26]. The base then leaves the oligonucleotide either protonated or as the neutral base, and the base loss is followed by a backbone cleavage. Studies have also been conducted on the fragmentation of oligonucleotides with a single metal ion attached [2733], and Wang et al. [34, 35] studied the collision induce dissociation (CID) of doubly negatively charged oligonucleotides with all the acidic phosphodiester protons exchanged against sodium ions. In these experiments the metal adducts of the [M 2H]2 ions were generated through electrospray ionization and the fragmentation upon collisional activation was studied with an ion trap. Recently, we published a study on the metastable decay of the sodium adducts and the sodium free singly charged deprotonated hexameric nucleotides 5=-d(TTXYTT) (X and Y are dC, dG, or dA) when formed in the MALDI process [12]. In this study, we found that the backbone cleavage of the deprotonated oligonucleotides is gradually quenched with increasing degree of sodiation and that quantitative quenching is generally achieved when four of the acidic protons have been exchanged against sodium ions. The base loss, on the other hand, increases with increasing sodiation, hence as the backbone cleavage decreases. Furthermore, when the sixth proton was exchanged against sodium and beyond that point we observed a relative destabilization of the pyrimidine bases compared with the purine bases. Our observations are concordant with the CID work of Wang et al. [34], which shows considerably higher stability for the metal adducts compared with the native [M 2H]2 ions and that the loss of pyrimidine bases becomes favorable compared with the purine base loss when the phosphodiester protons are exchanged against metal ions. Here we have extended our study to the post source decay (PSD) of protonated hexameric nucleotides 5=-d(TTXYTT) (X and Y are dC, dG, or dA). We study systematically the influence of the degree of sodiation, i.e., when the acidic protons are one by one exchanged against sodium ions, and we show that the role of sodium is not less significant for the protonated oligonucleotides. Similar to the case of the deprotonated oligonucleotides, we observe quenching of the classical w and a-B channels with increasing degree of sodiation of the protonated oligonucleotides, and we find the branching ratios between these two channels are highly dependent on the sequence and nature of the two central bases. In addition to the w and a-B channels, we observe a drastic and abrupt on/of-switching of different reaction channels as the degree of (...truncated)