7‐Deaza purine bases offer a higher ion stability in the analysis of dna by matrix‐assisted laser desorption/ionization mass spectrometry

Abstract

Oligodeoxynucleotides which contain 7-deaza analogues of the normal purine nucleotides have been synthesized both enzymatically and chemically. When subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis, the modified samples offer both higher stability and increased sensitivity compared to otherwise identical unmodified oligodeoxynucleotides. In view of these observations, models for the fragmentation of oligodeoxynucleotides in MALDI-MS with positive ion detection mode are presented. Additionally, the potential use of 7-deaza purine nucleotides in the MALDI-MS analysis of DNA sequencing reactions is discussed.

RNA fragmentation in MALDI mass spectrometry studied by H/D-exchange: Mechanisms of general applicability to nucleic acids

“…In a mechanism of neutral nucleobase loss earlier proposed for positive DNA-ions [5], which is in agreement with the data obtained here, the nucleobase leaves with the excess proton and does not abstract the nearby phosphate-OH hydrogen (Scheme 4). Early works on oligonucleotide fragmentation have shown that protonation destabilizes the nucleobase [22,[25][26][27][28], which substantiates the mechanism proposed in Scheme 4 and strongly indicates that the nucleobase carrying the excess positive charge is much more prone to cleavage than other nucleobases in the oligonucleotide. The excess proton on nucleobases of positive oligonucleotide ions allows cleavage of the N-glycosidic bond without separation of charge and formation of a likely energetically unfavorable zwitterion.…”

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

“…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.…”

“…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 ϩ H] ϩ ) in MALDI [24,41]. Thermodynamic arguments have been used to support the influence of the matrix on the fragmentation process.…”

DNA analysis by MALDI-TOF mass spectrometry

“…Many of the pioneers of DNA analysis devised a number of strategies to deal with these problems. Solutions included transcribing DNA into RNA, because RNA tended to show less depurination [Kirpekar et al, 1994], and using DNA with deazapurine bases [Kirpekar et al, 1995;Schneider and Chait, 1995]. A key observation made by Pieles et al [1993] was that adding ammonium ions during matrix preparation significantly reduced problems with sodium and potassium adducts.…”