Fragmentation pathway studies of oligonucleotides in matrix-assisted laser desorption/ionization mass spectrometry by charge tagging and H/D exchange

Chau-Wen Chou 0 1 3 4 Patrick A. Limbach 0 1 2 3 Richard B. Cole 0 1 3 0 Published online October 24, 2002 Address reprint requests to Dr. C.-W. Chou, Department of Chemistry, University of New Orleans , 2000 Lakeshore Drive, New Orleans, LA 70148, USA 1 Department of Chemistry, University of New Orleans , New Orleans, Louisiana, USA 2 Present address: Department of Chemistry, University of Cincinnati , Cincinnati, OH 45221 . This article is dedicated to Professor Peter Williams of Arizona State University in celebration of his 60th birthday 3 Department of Chemistry, Louisiana State University , Baton Rouge, Louisiana, USA 4 Also at the Department of Chemistry, University of New Orleans , New Orleans, LA 70148 The desorption and decompositions of synthesized oligonucleotides bearing fixed charge sites have been investigated by linear, delayed-extraction, reflecting and post-source decay mode matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In contrast to the conventional [M H] forms of unmodified molecules where a proton is likely attached to a nucleobase, here the charge is fixed at one of the termini. In this case the observed fragment ions always incorporate the charge-tag. H/D exchange experiments provide no evidence for intramolecular migration of protons on the phosphate backbone to initiate the fragmentation event. New unique pathways of proton migration from the ribose have been elucidated and are rationalized by a charge-remote fragmentation pathway. (J Am Soc Mass Spectrom 2002, 13, 1407-1417) 2002 American Society for Mass Spectrometry - Ttrometry has been an active area for many years he analysis of oligonucleotides using mass specdue to the demands for a rapid and accurate DNA sequencing technique as well as for the identification and characterization of modified oligonucleotides [1, 2]. Since Matrix-Assisted Laser Desorption/ Ionization (MALDI) has been introduced, [3, 4] much effort has been devoted to the development of oligonucleotide analysis by MALDI mass spectrometry. To date, Tang et al. [5] and Liu et al. [6] have reported the analysis of PCR products 500 to 600 bases using UV-MALDI (at 337/355 nm), and Hillenkamp and co-workers extended the upper limit of DNA and RNA analyses up to 2180 bases using IR-MALDI [7]. Applications such as analyzing short tandem repeats [8], gene-defect diseases [9 11], and genotyping of single nucleotide polymorphisms [12] have been attempted. Despite these successes, oligonucleotide analysis by MALDI-MS has been limited primarily by the low ion abundances of larger oligonucleotides. Routine analysis by UV-MALDI is still limited to approximately 50 bases in cases where good mass spectrometry performance (e.g., resolution and reproducibility) is of high importance [13]. The vast majority of the analyses of oligonucleotides by MALDI is limited to the identification and characterization of short chain oligonucleotides, such as antisense drugs [14, 15]. The most probable cause for the limited success at very high masses is that larger oligonucleotide ions are unstable and dissociate rapidly after the desorption/ ionization step. A large number of studies have been undertaken to better understand the fragmentation process [16 34]. Generally, the severity of fragmentation is matrix-dependent, and a matrix of choice is 3-hydroxypicolinic acid (3-HPA) [35, 36] (or the two component matrix-3HPA/picolinic acid [5]) because it results in a lesser amount of fragmentation and enables the analysis of larger oligonucleotides. The popular matrices for peptides and proteins, such as sinapinic acid (SA) instrument facility tend to produce significant fragmentation when used for oligonucleotide analysis. Despite the matrix dependency on the degree of decomposition, the types of observed fragment ions remain similar for almost all matrices that have been studied. The stability of molecular ions of oligodeoxynucleotides is highly dependent on their base composition. Studies of homopolymer and mixed bases of oligonucleotides reveal that fragmentation often occurs at the location of a guanosine, adenosine, or cytidine base (in decreasing order of fragmentation severity) [22, 37, 38]. The laser wavelength does not seem to be a determining factor. 3-HPA is an effective matrix for oligonucleotides at 266, 307, 337, and 355 nm. Even though IR photons have less energy, IR absorbing matrices such as succinic acid do not offer as much success as 3-HPA in the UV range [21, 39]. Several hypotheses have been proposed to rationalize observed fragmentation pathways, and all of the proposed mechanisms involve protonation of a nucleobase as the initiating step [22, 24, 39]. It has been proposed that proton transfer occurs from either the neighboring acidic phosphodiester groups to the nucleobase, thus forming short-lived zwitterionic intermediates, or from matrix ions to form stable zwitterions upon protonation of the nucleobase. The N-glycosidic bond is then polarized and weakened, which initiates base elimination leading to strand scission along the phosphodiester backbone. These hypotheses nicely explain the correlation of proton affinities (PA) of the four nucleobases to the observed fragmentation patterns: Gua (229.3 kcal/mol) Ade (225.3 kcal/mol) Cyt (227.0 kcal/mol) Thy (210.5 kcal/mol) [40]. Evidence supporting this ranking comes from the absence of prompt fragment ions from poly-(T)n desorbed from a variety of matrices in linear time-of-flight mass spectrometry. The 7-deaza purine analogs of guanosine and adenosine that are less acid labile in aqueous solutions compared to the native forms exhibit higher stability (as [M H] ) in MALDI [24, 41]. Thermodynamic arguments have been used to support the influence of the matrix on the fragmentation process. 3-HPA has the highest proton affinity (214.1214.5 kcal/mol) and therefore, when protonated, has the least capability to transfer that proton to an oligonucleotide compared to other common oligonucleotide matrices such as THAP (210.8 kcal/mol) and 2,5-DHBA (201208 kcal/mol) [25, 42 44]. To probe the fragmentation mechanisms in more detail, Hettich et al. used MALDI Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICR-MS) to characterize the structures of native and modified oligonucleotides through prompt and collision-induced dissociation fragmentation [45]. They elucidated the structures of fragment ions (metastable and stable prompt ions) and proposed fragmentation pathways. Gross et al. have investigated fragmentation pathways of metastable ions of dideoxynucleotide tetramers using post-source decay (PSD) and H/D exchange in a delayed extraction reflecting MALDI-TOF [28, 33]. They reported many fragment ions that are not observed in regular TOF-MS, such as y- and z-series ions. The H/D results revealed convincing details of the bond cleavages by clarifying hydrogen transfers. Wang et al. compared similarities and differences of T-rich oligonucleoti (...truncated)