Nucleic Acids Research, 1993, Vol. 21, No. 15 3347-3357 Ion stability of nucleic acids in infrared matrix-assisted laser desorption/ionization mass spectrometry E.Nordhoff, R.Cramer, M.Karas, F.Hillenkamp, F.Kirpekar1, K.Kristiansen1 and P.Roepstorffl Institute for Medical Physics and Biophysics, University of Munster, Robert-Koch-Strasse 31, 4400 Munster, Germany and 1Department of Molecular Biology, University of Odense, Campusvej 55, 5230 Odense M., Denmark Received May 28, 1993; Accepted June 21, 1993 ABSTRACT Matrix-assisted laser desorption/ionization mass suitable matrix of molecules having a strong absorption at the spectrometry (MALDI-MS) with infrared laser light of a laser wavelength and present in high molar excess over the wavelength of 2.94 zm has been used for the analysis analyte. This induces an efficient transfer ofthe laser-pulse energy of nucleic acids. Spectra of oligodeoxynucleotides up to the analyte and results in a soft desorption process. It is to 26 nucleotides, oligothymidylic acids up to 100 suggested that the matrix molecules also play a role in the nucleotides as well as different synthetic RNA ionization of the analyte molecules.9 Different lasers and oligomers and RNA transcripts up to 104 nucleotides wavelengths in the UV (mainly 266 nm, 337 nm and 355 nm) are presented. A main problem in the analysis of and IR (mainly 2.94 ,um, 2.79 ym and 10.6Am) have been used oligodeoxynucleotides was found to be related to the for this technique. Complex mixtures of proteins and peptides loss of bases. The stability of oligothymidylic acids as are routinely mass analysed. Carbohydrates'0, glycoproteins, opposed to oligodeoxynucleotides containing all four lipoproteins and lipids, as well as many other synthetic and natural bases indicates that the loss of bases is correlated with polymers11, are also amenable to MALDI-MS. In all of these A, C and G protonation which decreases the stability cases the key to successful analysis of a new class of analyte of the N-glycosidic bond. Experiments indicate that the molecules has been to find a specifically suited combination of breakage of the N-glycosidic bond probably occurs matrix and laser wavelength. during the desorption process due to proton transfer Provided that the right conditions for desorption of nucleic acids from the phosphodiester groups to the ionizable bases. of > 100 nucleotides could be found, rapid DNA sequencing RNA displayed a significantly higher stability in MALDI- would be possible by direct mass spectrometric analysis of the MS due to the presence of a 2'-OH group. Conse- Sanger reaction products'2, replacing the time consuming gel quently, signals of RNA transcripts with a length of up electrophoretic procedure. In many other fields of DNA research to 142 nucleotides could be detected by MALDI-MS. such as antisense DNA applications a quick and easy analysis Technical details of the method, including the of oligonucleotides of 10 to 30 bases would also be of high distribution of positive counterions on the phospho- interest. Typical modifications such as labelling with a diester backbone, the upper mass limit and mass fluorescence marker, the addition of a hydrophobic anchor or accuracy are discussed along with a number of the introduction of thiophosphate groups, etc. could be verified potential analytical applications. by a determination of the accurate molecular mass. Among the limited number of results, so far reported for MALDI-MS of oligonucleotides, are analyses of oligothymidylic acids, pd[T]8 to pd[T]30, and mixtures of them'3-21, of oligodeoxynucleotides INTRODUCTION (containing all four different bases)'3'17"19'20'22'23 and of RNA Whereas the matrix-assisted laser desorption/ionization mass samples. Karas et al. have reported spectra of tRNA'3,17,20, and spectrometry (MALDI-MS) has found broad applications for the Williams et al. have been able to obtain spectra of oligodeoxy- analysis of oligopeptides and proteins' -6, investigations on nucleotide samples of up to 60 nucleotides24. Recently, Becker nucleic acids are limited so far. Since its introduction in 1988 and co-workers25 have published mass spectra of oligodeoxy- by Karas and Hillenkampl, MALDI-MS has become an nucleotides up to a 67 mer. established method for desorbing ions of proteins with molecular In a recent publication20, we have compared oligodeoxy- weights up to 500 kDa. The determination of molecular masses nucleotide analysis with ultraviolet (UV)-MALDI with the 337 with an accuracy of up to 0.01 % is possible. Typical sample nm wavelength of a N2-Laser and a 2-aminobenzoic acid matrix amounts required are 1 pmol, but a sensitivity in the low to that of infrared (IR)-MALDI with the 2.94 /tm wavelength femtomol range can be achieved7'8. The key idea of this of an Er:YAG Laser and succinic acid as matrix and found the technique is to embed the macromolecules (the analyte) in a latter combination to give superior results. In the meantime, 3348 Nucleic Acids Research, 1993, Vol. 21, No. 15 further tests have shown that the combination of min: 100% B. 35-40 min: 100-0% B. 40-45 3-hydroxypicolinic acid as a matrix and a wavelength of 337 nm min: 100% A also yields very good results, in agreement with the finding of Flow rate: 50 A1/min other groups19'25. In this paper, results on the IR-MALDI analysis of a series of synthetic oligodeoxynucleotides in the size Oligoribonucleotides.Synthetic oligoribonucleotides as well as range from 12 to 26 nucleotides as well as for some synthetic RNA oligomers from in vitro RNA polymerase transcription RNA oligomers up to 19 nucleotides and RNA-transcripts up to reactions and also from limited degradation ofpolyuridylic acids 104 nucleotides are reported. The aim of the study was to find were used. optimal conditions for sample preparation and to prove the feasibility of IR-MALDI-MS for nucleic acid analysis. 1. Synthetic RNA. The synthetic oligoribonucleotides were synthesized and HPLC purified by Dr. Jens Peter Fiirste, Freie Universitiit of Berlin, Germany. These samples were used as EXPERIMENTAL supplied. Instruments 2. In vitro transcripts. Aliquots of the plasmid pBluescript KS+ The mass spectrometer used for the analysis of the were linearized with the restriction endonucleases HindI, EcoRI oligonucleotides is a home built reflectron time-of-flight and NotI, which will generate transcripts of 55 nucleotides, 67 instrument (RTOF, Fig. 1)20. The system is equipped with an nucleotides, and 104 nucleotides, respectively, in an in vitro electro optically Q-switched Er:YAG laser (1-2-3 Schwartz transcription system using T3 RNA polymerase. Also, the Electro-Optics, Orlando, FL, USA) emitting 150 ns pulses at plasmid pBluescript SK+ was digested with the restriction a wavelength of 2.94 gm. The laser beam is focused onto the enzymes Hinfl, which will generate a transcript of 142 nucleotides sample surface under 450 to a spot diameter of ca. 100 /km. The in the above mentioned transcription system. The plasmids were laser irradiance varied between 106 and 107 W/cm2. The ions subsequently extracted with phenol/chloroform and precipitated are accelerated to an energy of 12 keV and detected by a with ethanol/Na-acetate. secondary electron multiplier (EMI 9643) equipped with a In vitro transcription was performed in 100 Al containing separately mounted dynode for ion-ion conversion. An 40 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 2 mM spermidine, instrumental mass resolution at up to 1000 (m/Am, Am: full width 10 mM NaCl, 10 mM DTT, 100 units RNAsin (Promega Biotec), at the half maximum (FWHM) of the peak) can be obtained with 0.5 mM each of ATP, CTP, GTP and UTP, 5 ytg of linearized this instrument for ions below ca. 5000-6000 Da under optimal plasmid and 50 units of T3 RNA polymerase (Stratagene). The conditions. Above 10,000 Da, the mass resolution is reduced to reaction was incubated at 37°C for one hour, another aliquot of a value of < 200 due to both the ion detection process (conversion 50 units T3 RNA polymerase was added and incubation was of the analyte ion to secondary ions) and the ion formation continued for one more hour. The reaction mixtures were process. The analog detector signal is digitized by a transient extracted once with phenol/chloroform, precipitated with recorder (LeCroy 9400) at time intervals of 10 or 20 ns. Further ethanol/NH4-acetate and dissolved in 15 Al of 7 M urea. data processing was done on a PC-AT using in-house generated The transcripts were electrophoresed through a 5% software. Generally, single shot spectra clearly show the polyacrylamide sequencing gel containing 8 M urea and visualised molecular ion signals. To improve the signal-to-noise ratio, 10 on a fluorescent thin layer chromatographic plate under 254 nm to 20 single shot spectra were usually accumulated and averaged. irradiation and subsequently cut out ofthe gel. RNA was extracted from the gel plug by gentle shaking in 200 Al 10 mM Tris-HCl Sample preparation (pH 8.0), 20 mM EDTA, 0.5% SDS and 50 jig/ml proteinase Matrix. The matrix, succinic acid, was dissolved in ultra pure K at 30°C for 16 hours; the gel plug was washed with 100 IlI water to a concentration of ca. 50 g/l and then carefully desalted TE-buffer and the pooled liquid was extracted once with on an acid (0.1 M HCI (p.a.) solution) activated cation exchange chloroform. RNA was precipitated twice with 2 M NH4-acetate column (cation exchange polymer: BioRad, 5OW-X8, mesh size and 3 volumes of ethanol. 100-200 ltm). In addition to the preparative transcription reactions, analytical Subsequently, the purified matrix solution was diluted to a reactions of 1/10 volume supplemented with 8 AM of concentration of 20 g/l, aliquoted in 50 ,ul portions and stored [a-32P]-UTP (800 Ci/mmol) were made and purified in parallel.