Particle Beam Glow Discharge Mass Spectrometry: Spectral Characteristics of Nucleobases

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RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2003; 17: 1749–1758 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1117 Particle beam glow discharge mass spectrometry: spectral characteristics of nucleobases

W. Clay Davis, Jacob L. Venzie, Bert Willis, R. Lane Coffee Jr., Dev P. Arya and R. Kenneth Marcus* Department of Chemistry, Howard L. Hunter Chemical Laboratory, Clemson University, Clemson, SC 29634-0973, USA Received 22 October 2002; Revised 1 June 2003; Accepted 1 June 2003

Use of a particle beam glow discharge (PB-GD) source for mass spectrometric determinations of deoxy- and ribonucleosides and nucleotides is described. Use of this combination of sample introduction and ion source decouples the vaporization and ionization steps, leading to very simple spectral structure. The mass spectra of these compounds are EI-like in nature, with clearly identified molecular ions and fragmentation patterns that are easily rationalized. The PB-GDMS combination can be operated in a flow injection mode wherein the analyte is injected directly into the solvent flow, or can also be coupled to a high-performance liquid chromatography (HPLC) system allowing LC/MS analysis of mixtures. Mass spectra obtained for nucleic acid bases, nucleosides, and nucleotides are readily obtained with injections of low-nanomole quantities. Representative PB-GDMS spectra for deoxy- and ribonucleosides, nucleotides, and mixed-base oligonucleotides are presented to demonstrate the capabilities of the GD source. Characteristic fragmentation peaks from the spectra of adenine, cytosine, guanine, and thymine were identified in 22-base sequences of single-stranded DNA. The PB-GD source is capable of producing spectra that may be used to identify the individual bases present in mixed-base DNA and RNA fragments. Copyright # 2003 John Wiley & Sons, Ltd.

Mass spectrometry is an important tool in the analysis of bio- chemical derivatization of degraded proteins and polynu- molecules because it provides both the identities and abun- cleotides (e.g., via Edman or exonuclease chemistries) is dances from the mass spectra (molecular ion and required to form volatile species that can be analyzed by fragmentation) of the molecule. Far and away the most com- electron ionization. It would be desirable, though, to have mon ionization sources for the introduction of large biomole- available sample introduction/ionization schemes that allow cules for mass spectrometric detection are electrospray analysis of in-solution species, without derivatization, with ionization (ESI) and matrix-assisted laser desorption/ioniza- mass spectra that exhibit fragmentation patterns that are tion (MALDI).1–6 ESI allows for multiply charged molecular similar in structure to those provided by EI in that subunits ions to be generated from analytes such as peptides, proteins, are readily identified. and nucleic acids, with minimal analyte fragmentation. ESI While Sanger dideoxy chemistry is by far the most widely has an added advantage of being readily coupled directly to used detection technique for sequencing DNA, mass spectro- a variety of chromatographic and electrophoretic separation metry is becoming more widely utilized as a detection techniques for purification or separation of complex mixtures. technique for sequencing small DNA fragments. The use of The MALDI source also has the ability to generate ions of large MALDI time-of-flight mass spectrometry (TOFMS) has been analytes such as proteins and peptides without significant explored widely in sequencing.7–10 Recently, modified base fragmentation, although signals due to matrix ions render detection with isotope ratio mass spectrometry11 (IRMS), and the lower mass range too complex for ready extraction of ana- the use of ESI-TOF for the detection of triphosphate forms of lytical information. MALDI has also been used in liquid chro- the deoxynucleobases,1 have been reported. matography for off-line determination of collected fractions.2 Glow discharge (GD) devices are widely used for In order to obtain mass spectral information of small analytical applications in the area of metals, alloys, and molecule segments/components (e.g., single amino acids, semiconductor materials.12 Such applications are the result of nucleic acids) of larger biomolecules, supplemental frag- the efficient and easily controlled atomization, excitation, and mentation is required for both ESI and MALDI. Alternatively, ionization processes occurring in these sources. The combination of cathodic sputtering (atomization) and gas-phase *Correspondence to: R. K. Marcus, Department of Chemistry, collisions for both the excitation and ionization processes Howard L. Hunter Chemical Laboratory, Clemson University, produces a powerful tool for direct solids analysis. In Clemson, SC 29634-0973, USA. E-mail: [email protected] addition, the GD plasma presents a number of favorable Contract/grant sponsor: Protasis Corporation. qualities relative to the analysis of samples originating from

Copyright # 2003 John Wiley & Sons, Ltd. 1750 W. C. Davis et al. the solution and gas phase. Gaseous sample introduction molecules/components such as single nucleobases and directly into the discharge is straightforward and allows for oligonucleotides may be a useful addition to the mass the analysis of both organic and organometallic species by spectrometry portfolio. atomic emission and mass spectrometry.13–17 The extension We describe here a demonstration of the PB-GDMS of the GD techniques to the direct analysis of liquid samples, approach to the analysis and identification of individual however, is hindered by the inability of the low-pressure/ bases in both deoxy- and ribonucleosides and nucleotides of low-temperature plasma to effectively desolvate the ana- adenine (A), cytosine (C), guanine (G), thymine (T) and uracil lyte.18 In addition, the presence of solvent vapors (e.g., water) (U), as well as oligonucleotides. Easily interpreted spectra greatly affects the plasma energetics and leads to spectral from both the single nucleosides and nucleotides can be complexity due to adduct formation. utilized to identify the constituent bases of mixed-base The desire to make the GD source a multifaceted mass oligonucleotides with this technique. The relative simplicity spectrometric analysis tool has led researchers to explore the of the nucleobase spectra could be envisioned to be of utility possibilities of using the source for analysis of both inorganic in studying single-base modifications such as deamination and organic solutions. To this end, Marcus and co-workers chemistries. have demonstrated the use of the particle beam (PB) as a means of introducing liquid-phase samples into GD EXPERIMENTAL sources.19–24 The PB interface, originally applied in the MAGIC/LC/MS approach of Willoughby and Browner,25 Instrumentation provides a means of introducing LC effluents into low- The basic components of the liquid chromatography particle pressure sources (i.e., electron impact and chemical ioniza- beam glow discharge mass spectrometry (LC/PB-GDMS) tion) while maintaining chromatographic characteristics instrument are shown in Fig. 1.23 The apparatus is comprised such as retention and elution quality.26,27 Other advantages principally of an ABB Extrel (Pittsburgh, PA, USA) Bench- of the PB interface are its mechanical/operational simplicity, markTM LC/MS quadrupole mass spectrometer system and compatibility with a wide range of solvent polarities and flow a Waters (Milford, MA, USA) model 510 HPLC pump, rates, as well as its high efficiency in solvent removal. As employing a Rheodyne (Rohnert Park, CA, USA) model such, the PB interface is well suited for the introduction of 7725i injector with a 50 mL sample loop used throughout the solution-phase analytes into GD sources. The coupling of the work. Experiments described here were performed in the dis- particle beam interface to a glow discharge mass spectro- crete (flow) injection operating mode. The output of the flui- metry (PB-GDMS) ion source produces a high level of dic system is coupled to the nebulizer of the PB apparatus by a information content for both molecular and elemental species 45 cm segment of 0.5 mm i.d. PEEK tubing. in solution analyses.23,24 Specifically, the mass spectra of a The PB interface (ThermabeamTM, Extrel, Pittsburgh, PA, wide variety of compounds (e.g., selenoamino acids, hor- USA) includes a thermoconcentric nebulizer to generate a mones, and organometallics) are very EI-like in nature, finely dispersed aerosol, a metal spray chamber for desolva- yielding molecular ions and easily identified fragmentation tion, and a two-stage momentum separator which removes patterns. The extension of these capabilities to small residual solvent vapor. Specifically, the liquid sample is

Figure 1. Diagrammatic representation of the basic components of the particle beam/ glow discharge mass spectrometry (PB-GDMS) apparatus.

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 1749–1758 PB-GD-MS of nucleobases and nucleotides 1751 introduced via the HPLC pump into a fused-silica capillary 1.95 nmol injected; Integrated DNA Technologies, Coralville, (75 mm i.d.) mounted within a stainless steel tube (1.6 mm IA, USA) were also prepared in Milli-Q water. o.d.). A DC potential difference placed across the stainless- steel tubing results in resistive heating along the tube, providing a thermal component to the aerosol formation. RESULTS AND DISCUSSION Helium is introduced as an annular sheath gas to improve Identification of single bases in mixed-base biomolecular heat conduction and break up the liquid at the capillary tip fragments is required for sequence elucidation of both RNA (i.e., pneumatic nebulization). The resulting aerosol is and DNA, regardless of the actual methodology. As such, introduced directly into a heated (1008C) stainless-steel sequence- or base-specific fragments in the <500 Da mass desolvation chamber. The separation of the analyte particles range provide this important information. Traditional ESI from the aerosol mixture occurs as it passes through the and MALDI sources provide little or no fragmentation and differentially pumped momentum separator, which skims therefore must include a secondary fragmentation step (i.e., off the low-mass nebulizer gas and solvent molecules. The collisional dissociation) in the mass analysis of oligonucleo- resulting beam of desolvated analyte particles (1–10 mm dia.) tides.5,28–31 Because of its bimodal ionization mechanism is introduced into the heated (2808C) discharge cell, (i.e., EI and Penning),12 the glow discharge source is soft vaporized from the probe surface, and subsequently ionized enough to provide molecular weight ions while still provid- within the plasma. ing sufficient fragmentation for unambiguous identification The mass spectrometric analyses were performed using a of individual nucleobases, nucleosides, and nucleotides. commercial particle-beam quadrupole (2000 Da mass range) instrument (BenchmarkTM, Extrel). The only substantive modification of the commercial system was the replacement Nucleosides of the conventional electron impact source volume with a The deoxy- and ribonucleosides and the nucleotides served glow discharge source of the same cell geometry.23 The glow as benchmarks for the analysis of the oligonucleotides. discharge source uses a 12.5 mm diameter direct insertion Figures 2 (a) and 2(b) show the mass spectra of 50 mM solu- probe (DIP), designed for GDMS analysis of metallic tions (0.6 mg) of 20-deoxyadenosine and 20-deoxythymidine, samples, which is inserted through the ‘solids probe’ inlet respectively, as examples of the spectral composition of the to the mass spectrometer chamber and into the ion volume. In four DNA nucleosides. The [MþH]þ ions of both nucleosides, this way, a 4 mm diameter copper target (i.e., the cathode in along with the protonated bases, have been identified and the GD plasma source) is introduced perpendicular to the labeled. The ionized sugar fragment is also present in the path of the incoming analyte particles. Particles impinging spectra. Figure 2(a) demonstrates a typical fragmentation on the cathode surface are vaporized (1508C) and diffuse pattern for nucleobases containing an amine (NH2) group into the plasma negative glow region. It is important to note on the base (adenosine, cytosine, and guanine). Specifically, that this vaporization process is sufficiently energetic to a fragment ion representing the nucleobase minus 16 Da þ þ dissociate the desolvated particles to molecular form, but no ([B–16] , i.e., [MþH–NH3] ) is observed. A peak corre- evidence of pyrolysis has been seen. Analyte species ionized sponding to the loss of ammonia is detected throughout through collision with electrons or metastable Ar species this work for all of the bases with this functionality, although (Penning ionization) are sampled through a 1 mm exit these are not always labeled in the spectra. For comparison, aperture to the quadrupole mass analyzer. Mass spectra Fig. 2(c) presents the EI spectrum of 20-deoxyadenosine.32 were recorded in both total ion chromatogram (TIC) and As can be seen, there is good agreement in the component single ion monitoring (SIM) modes of data acquisition using peaks in the PB-GDMS (Fig. 2(a)) and EI spectra of this com- the IonstationTM (ABB-Extrel) data system software. The data pound. The GD source yields a higher abundance of the were exported and processed by SigmaPlot 8.0 (SPSS Science, [MþH]þ ion, with approximately the same fractions of other Chicago, IL, USA). The glow discharge is powered by a fragments. Of particular note is the absence of the ammonia Spellman high voltage DC supply (model RHR5N50; Plain- loss ion in the EI spectrum. The mass spectra of other DNA view, NY, USA), operating in a constant current mode nucleosides show essentially the same spectral composition. (1 mA at 2000 V). A discharge gas of UHP Ar (National Figures 3(a) and 3(b) show the PB-GDMS mass spectra of Welders, Charlotte, NC, USA) was used throughout the 50 mM solutions (0.7 mg) of adenosine and guanosine as study. examples of the spectral composition of the four RNA nucleosides. Identification of the base with the loss of the Samples amine group (m/z 136) and the sugar (m/z 134) is readily Stock solutions (50 mM, 2.50 nmol injected) of the nucleosides accomplished because of the easily interpreted spectra. (20-deoxyadenine, 20-deoxycytosine, 20-deoxyguanine, 50- Comparison between the PB-GDMS adenosine spectrum deoxythymine, adenine, cytosine, guanine, uracil) and and the corresponding one from an EI library32 (Fig. 3(c)) nucleotides (triphosphate sodium salts of deoxyadenine, shows very good correlation in terms of component species, deoxycytosine, deoxyguanine, and deoxythymine and with the GD-derived spectrum again showing a greater monophosphate salts of adenine, cytosine, guanine, uracil; propensity to yield [MþH]þ ions. In this case, the loss of the Sigma-Aldrich, Milwaukee, WI, USA) were prepared in amine functionality of the base is seen in the EI spectrum. It is Milli-Q (Millipore, Milford, MA, USA) water. Stock solutions of particular interest that both the deoxy- and ribonucleo- of 22mer deoxyoligonucleotides 14A8G (25.7 mM, 1.29 nmol sides yield ions representative of the molecular species and injected) and its complement strand 14T8C (38.9 mM, bases that are both in the form of radical cations (Mþ.) and

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 1749–1758 1752 W. C. Davis et al. protonated species [MþH]þ. Although the prominent peak at reactions. The different responses observed here in the glow m/z 237 in the EI spectrum (likely reﬂecting the loss of the discharge spectra for DNA and RNA species must be due to methoxy moiety) is also seen in the GD spectrum, its intensity the differing chemistries of these compounds that are in fact in the GD spectrum is very low. Typically, GD-generated non-protonated at neutral pH values. All previous PB-GD mass spectra of organic compounds yield classical EI type studies were performed on molecules that were not proto- spectra [Mþ.],23,24 while APCI techniques give protonated nated or were deprotonated in pH-neutral solutions. It is molecules ([MþH]þ ions) through gas-phase acid/base interesting to note that the EI spectra of these species

Figure 2. Mass spectra of 50 mM solutions of (a) 20-deoxyadenosine and (b) 20-deoxythymidine. Solution ﬂow rate 0.6 mL/min, 50 mL injection, discharge pressure 480 mTorr Ar, discharge current 1.0 mA. (c) EI mass spectrum of 20-deoxyadenosine.

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 1749–1758 PB-GD-MS of nucleobases and nucleotides 1753

Figure 2. Continued

(Figs. 2(c) and 3(c)) show some protonation of the bases as dine triphosphate DNA nucleotides, respectively. The spec- well, likely a reﬂection of residual water. tra show the same nucleoside peaks seen in the previous spectra, along with protonated and non-protonated forms of the molecular species (e.g., [dATP]þ. and [dATPþH]þ) and phos- þ Nucleotides phate ions ([H3PO3] ). Each of the peaks has been identiﬁed, Figures 4(a) and 4(b) show the mass spectra of 50 mM solu- including the molecular ions (i.e., the triphosphates), dipho- tions (1.2 mg) of the 20-deoxyadenosine and 20-deoxythymi- sphate, monophosphate, nucleosides, bases, sugars, and

Figure 3. Mass spectra of 50 mM solutions of (a) adenosine and (b) guanosine. Solution ﬂow rate 0.6 mL/min, 50 mL injection, discharge pressure 480 mTorr Ar, discharge current 1.0 mA. (c) EI mass spectrum of adenosine.

Figure 3. Continued phosphate groups, as well as other fragments. Those peaks ination of the amine group from cytosine seen in Fig. 5(a) pro- not labeled include residual hydrocarbon-like peaks likely vides positive differentiation between the two isobaric arising from the sugar moiety. Figures 5(a) and 5(b) show nucleobases. This is important because it allows the use of a the mass spectra of 50 mM solutions (0.8 mg) of the cytosine simple and predicable fragmentation pattern to discriminate and uridine monophosphate RNA nucleotides. The RNA between two bases with excocyclic amine and carbonyl spectra show similar fragmentation patterns to those of the groups of the same mass without the use of collisional disso- DNA nucleotides. While the RNA nucleosides of cytosine ciation. The ability to distinguish between these functional- and uridine have the same nominal molecular mass, the elim- ities would be useful in the monitoring of de novo

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 1749–1758 PB-GD-MS of nucleobases and nucleotides 1755

Figure 4. Mass spectra of 50 mM solutions of (a) dATP and (b) dTTP. Solution flow rate 0.6 mL/min, 50 mL injection, discharge pressure 480 mTorr Ar, discharge current 1.0 mA. biosynthesis of purine and pyrimidine nucleotides as well as Oligonucleotides studies of base-specific modifications. The mass spectra of the The mass spectrum of the mixed-base 22mer deoxyoligonu- cytosine and guanine DNA nucleotides and adenosine cleotide 14A8G (25.7 mM, 9 mg), presented in Fig. 6(a), and guanine RNA nucleotides (not shown) reflect the same clearly indicates the presence of the A and G bases and corre- general fragmentation patterns as those shown in Figs. 4 sponding phosphate residues. This spectrum demonstrates and 5. the ability of the GD source to provide singly charged ions

Figure 5. Mass spectra of 50 mM solutions of (a) CMP and (b) UMP. Solution flow rate 0.6 mL/min, 50 mL injection, discharge pressure 480 mTorr Ar, discharge current 1.0 mA. that can be utilized for the identification of individual bases (38.9 mM, 13 mg). The peaks corresponding to the individual present in mixed-base DNA. It should be noted that when the bases T and C have been identified. At first glance, the respec- amine group is fragmented off the deoxyguanine base, the tive peak heights for the molecular ions reflect the relative resultant fragment has the same mass (135 Da) as the deoxya- nucleobase compositions in the respective strands. Single denine base. Accordingly, some of the signal at that particular ion monitoring (SIM) was used to determine whether or not m/z value can be attributed to both compounds. Figure 6(b) the intensity ratios of the individual bases corresponded to shows the spectrum of the complementary strand 14T8C the percentages of each base present in each strand. The

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 1749–1758 PB-GD-MS of nucleobases and nucleotides 1757

Figure 6. Mass spectra of (a) 25.7 mM solution of 14A8G and (b) 38.9 mM solution of 14T8C of 22mer ssDNA. Solution ﬂow rate 0.6 mL/min, 50 mL injection, discharge pressure 480 mTorr Ar, discharge current 1.0 mA.

respective protonated forms of the bases, i.e., [BþH]þ m/z 136 (A). Based on the peak heights in the spectrum shown in (A), m/z 112 (C), m/z 152 (G), and m/z 127 (T), were monitored Fig. 6(a), the ﬁrst strand has a guanine content 61% of that because of their much higher intensity relative to those of the of adenine, while the SIM monitoring produced a value of molecular radical cations. Given the ratio of bases (8:14), the 53%. For the complementary strand, the peak heights of intensity of the peak corresponding to the 8 G bases in each Fig. 6(b) yield a cytidine fraction that is 66% that of the thymi- strand should be 57% of that of the major strand component dine content, and the integrated SIM intensities of replicate

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 1749–1758 1758 W. C. Davis et al. injections yielded the more correct value of 54%. The repro- mass spectrometry, for example, in the identification of DNA ducibility of the SIM peak areas was better than 4% RSD for damage products. triplicate injections of the respective strands. One obvious question pertaining to the analysis of oligonucleotides by PB-GDMS is the presence of signals Acknowledgements representing multiple nucleotides. In fact, there are clear Financial support from the Protasis Corporation is gratefully mass spectrometric signatures for ions of the 2mers and acknowledged. D.P.A. would also like to acknowledge sup- 3mers of T and C, although precise identification in this port from NSF CAREER award CHE-0134932. region of the mass spectra is difficult because of the limited mass calibration quality of the instrument when using heptacosafluorotributylamine, which only has characteristic REFERENCES peaks up to m/z 502, as the mass calibrant. The incorporation 1. Null AP, Nepomuceno AI, Muddiman DC. Anal. Chem. of a PB-GD source on a higher mass range and transmission 2003; 75: 1331. analyzer, such as a time-of-flight mass spectrometer, is 2. Doneanu CE, Griffin DA, Barofsky EL, Barofsky DF. J. Am. Soc. Mass Spectrom. 2001; 12: 1205. envisioned to provide greater levels of information for larger 3. Gupta R, Kapur A, Beck JL, Sheil MM. Rapid Commun. Mass strands of DNA, RNA, and other biomolecules, though the Spectrom. 2001; 15: 2472. practical upper mass limit is uncertain. 4. Huang YP, Konse T, Mechref Y, Novotny MV. Rapid Com- mun. 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