Influence of co-matrix proton affinity on oligonucleotide ion stability in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In this article we investigated the role organic base co-matrices play in reducing oligonucleotide fragmentation during analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The organic base co-matrix plays an important role in influencing the gas-phase behavior of desorbed oligonucleotides. No correlation was found between the solution pH values and the molecular ion stability of two model oligonucleotides. Instead, a direct correlation between the co-matrix proton affinity and the oligonucleotide molecular ion stability is seen. A co-matrix whose proton affinity is close to or greater than the proton affinity of the nucleobases can serve as a “proton sink. ” We propose that upon laser desorption/ionization, the co-matrix competes with the nucleobases of the oligonucleotide for additional protons from the matrix. When a co-matrix such as triethylamine is added, the co-matrix, rather than the oligonucleotide nucleobases, is the preferred site of proton transfer from the matrix. Titration of standard oligonucleotide matrices with several co-matrices of differing proton affinity demonstrates that the co-matrix mole fraction is an important factor in oligonucleotide molecular ion stability. When the mole fraction of the co-matrix approaches that of the matrix, nearly complete elimination of oligonucleotide fragmentation is seen. Control experiments utilizing pyridine, a co-matrix whose proton affinity is less than that of thymine or the phosphodiester backbone, demonstrate that the co-matrix plays an active role in oligonucleotide stabilization. Information on matrix:co-matrix interactions with these analytes should facilitate improvements in MALDI-MS of oligonucleotides.