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Comparison of Rhodamine Dyes as Matrices for ~a t n x -~i s t~ Laser D~o r p t i o n~o n~a t i o n Mass Spectrometry

Matrix-assisted laser desorptionlionization mass spectrometry has been used to mass analyze synthetic oligomers of ply-T ranging in size from 20 to 100 nucleotides. Both positively-and negatively-charged parent molecular ions were observed.Matrix-assisted laser desorptionhonization (MALDI) has been quite successfully used to detect very large protein molecules.'-3 However, the success of using MALDI on oligodeoxyribonucleotides is somewhat limited. Negative-ion spectra of d ( In this work, we report the observation of both positive-and negative-ion spectra of large oligomers of polydeoxyribothymidylic acid. The mechanism of producing large oligomer ions and the potential for fast sequencing of DNA by laser mass spectrometry is briefly discussed. EXPERIMENTALA Nd-YAG laser (Spectra Physics DCR2, Mountain View, CA, USA) capable of delivering four wavelengths (i.e., 1064 nm, 532 nm, 353 nm, and 266 nm) was used for laser ablation. A linear time-of-flight mass spectrometer (TOF) was used to obtain mass spectra. A schematic diagram is shown in Fig. 1 used in this work were made synthetically. Single-stranded oligonucleotides were synthesized using phosphoramidite chemistry on ABI (Applied Biosystems Inc., Foster City, CA, USA) model 392 automated DNA/RNA synthesizer at the University of Tennessee Molecular Biology Core Facility. The DNA samples were all constructed on a solid-phasecontrolled pore glass (CPG) from 3' to 5' using dT{dA, dG, dC, dI, dU}-CE phosphoramidites: 5'-dimethoxytrityl-2'-deoxythymidine, 3'-[(2-cyanoethyl) A full trityl assay was carried out to assess the coupling efficiency and the quality of each synthesis at 497 nm on the stepwise collection of dimethoxytrityl (DMT) cations. The oligomers (after being cleaved off the glass support using 30% ammonium hydroxide) were then subjected to 10 h of deprotection at 55 "C. All DNAs were activated at the 5'-end and D-ribose backbone by removing 5'-DMT and betacyanoethyl protecting groups as conventionally required by the molecular biological applications. Cieaved oiigomers were then desaIted and purified of failure sequences through extractions with n-butanol. This method obviates the need for prior NH3 removal, and results in a high yield of oligonucleotides suitable for use in DNA sequencing, PCR amplification and gel shift analysis of protein-DNA interactions.'6 Purified oligodeoxynucleotides were purity-scanned from 220 to 300 nm and quantified at OD-260 nm using a UV spectrophotometer before lyophilization. The samples were prepared by mixing an aqueous analyte solution with an aqueous matrix solution with selected mole ratios of matrix to analyte. About 30pmol of analyte was put onto a target plate with a spot size of -6mm. The

Various isomer matrices were used for laser-desorption ionization of mixtures of oligomers. It was found that the efficiency of production of oligomer ions changed drastically when different isomers were used as matrices. For selected matrix materials, parent oligomer ions with sizes up to 64 bases were observed.

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