synthesis of electrophore-labeled oligonucleotides and characterization by matrix-assisted laser...

JOURNAL OF MASS SPECTROMETRY, VOL. 31, 661-668 (1996)

Synthesis of Electrophore-labeled Oligonucleotides and Characterization by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry?

Phillip F. Britt, Gregory B. Hurst* and Michelle V. Buchanan Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA

Recent work to apply mass spectrometric methods to DNA analysis has led to the attachment of an electrophore to an oligonucleotide primer, with the purpose of investigating whether the advantages of electron capture ionization (increased ionization efficiency, reduced fragmentation) could be extended to larger molecules, such as Sanger sequence ladders. The stability of the electrophore-modified primers under conditions encountered during matrix- assisted laser desorption/ionization mass spectrometry (MALDI-MS) was investigated. Four different electrophore labels were successfully attached to the 5' terminus of a 17-base, single-stranded oligodeoxyribonucleotide sequencing primer. The attached electrophore tags are robust under conditions used for sample preparation and MALDI- MS, and little or no fragmentation resulting from loss of the electrophore was observed. While no sensitivity enhancement was observed for the electrophore-labeled DNA, mass spectrometric conditions are discussed under which the electrophore labels could enhance the detection of DNA sequencing ladders.

KEYWORDS: electrophores; oligonucleotides; derivatization; MALDI ; sequencing

INTRODUCTION

In order to speed up sequencing'.' and other DNA analyses4 that yield information encoded as a series of DNA fragments of different molecular weights, strategies based on matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI- MS)4,5 are being d e ~ e l o p e d ~ - ~ as alternatives to the lengthy gel electrophoresis separation and detection methods that are currently used. Because the matrix absorbs the bulk of the energy, intact ions are typically observed, particularly for proteins. Oligonucleotides, especially larger species, are more prone to fragmentation, although this problem can be addressed to some degree through the choice of the matrix compound.'' Ignoring sample preparation, signal averaging and data handling, a complete mass spectrum can be obtained by MALDI-MS in less than 1 ms, which potentially is a considerable time savings over electrophoretic methods.6 Sequencing by mass spectrometry also offers the possibility of improved accuracy over gels. For instance, phenomena such as base stacking will not affect mass spectrometric measurements.

One challenge in applying MALDI-MS to DNA detection is that the sensitivity, although excellent for small oligonucleotides (up to 30 bases), falls off drasti- cally with increasing molecular weight.6~ii*i2 Before

* Author to whom correspondence should be addressed. t Presented in part at the 43rd ASMS Conference on Mass Spec-

trometry and Allied Topics, Atlanta, GA, May 21-26,1995.

DNA can be detected by mass spectrometry, two processes must occur, preferably with neither causing fragmentation: desorption (transfer of the DNA from the solid phase into the gas phase) and ionization (production of a net charge on the gas-phase DNA). It has been shown that double-stranded DNA containing 622 base pairsI3 and single-stranded DNA containing an average of 400 nucleotide~'~ can be desorbed, trans- ported across a vacuum and collected intact on a surface. However, in these experiments, the large DNA species were not detected as ions in the gas phase, but by subsequent electrophoretic analysis of the collected desorbate. If these larger DNA species could be not only desorbed but also efficiently ionized in the vacuum, mass spectrometric detection would be greatly facili- tated. Therefore, we are investigating methods for enhancing ionization efficiency in order to improve MALDI-MS sensitivity.

Our first approach has been to derivatize DNA with an electrophore (a group with a high electron affinity' 5 ) ,

based on the observation that electrons are liberated from the matrix and substrate during the desorption/ ionization laser pulse.16 Electron capture by a neutral molecule produces a negative ion, which can be subjected to mass analysis in the mass spectrometer. Elec- tron capture ionization is widely used for small molecules with high electron affinities, and the sensitivity obtained can be as much as two to three orders of magnitude greater than that obtained using conventional electron impact i~nization. '~ Because electron capture can result in the deposition of relatively low excess energy in the negative analyte ion, fragmentation

CCC 1076-5174/96/060661-08 0 1996 by John Wiley & Sons, Ltd.

Received 8 January 1996 Accepted 7 March 1996

662 P. F. BRIlT, G. B. HURST AND M. V. BUCHANAN

is less likely to occur, and intact molecular ions can be observed with greater relative abundance than in other more energetic ionization methods. Derivatization of nucleic acid monomers with pentafluorobenzyl bromide (C,F,CH,Br) has been described for subsequent detection by gas chromatography/electron capture detection and by negative ion chemical ionization mass spectrometry." Oligonucleotides have also been labeled with 'release-tag' electrophores which are detached from the oligonucleotide prior to gas chromatographic separation and dete~ti0n.l~ Fatty acids, oligosaccharides and related compounds have been derivatized with the pentafluorobenzyl group to enhance sensitivity in negative ion chemical ionization mass In this report on the first phase of our investigation into applying mass spectrometry to DNA analysis, we discuss a general synthetic methodology for the attachment of an electrophore to an oligonucleotide primer, the stability of the electrophore-labeled oligonucleotide during MALDI-MS analysis and the potential of the electrophores for enhancing DNA detection by electron capture.

EXPERIMENTAL

General

All reagents, of HPLC or reagent grade, were obtained from commercial suppliers and used without further purification unless stated otherwise. Dimethyl- formamide (DMF) and diisopropylethylamine were dried over 4A sieves before use. Absorbance spectra were measured on a Cary 4E spectrophotometer (Varian). Gas chromatography was performed on a Hewlett-Packard 5890 Series I1 gas chromatograph equipped with a J&W 30 m x 0.25 mm i.d. DB-1 methylsilicone capillary column (0.25 pm film thickness). GC/MS analysis was performed on a Hewlett-Packard 5792/5890 Series I1 GC/MS system at 70 eV with a column identical with that used for GC analysis. Reversed-phase HPLC was performed on a Waters 600E multisolvent delivery system with a Waters 996 photodiode-array detector (1230-450 nm) equipped with a Hamilton PRP-1 column (250 x 4.1 mm i.d.). A linear gradient of 0.1 M aqueous tri- ethylammonium acetate-acetonitrile (95 : 5) against acetonitrile was used, ranging from 0 to 75% over 40 min at 1 ml min-'.

Electrophore synthesis

p-Nitrophenyl isothiocyanate (98%) was obtained from Aldrich Chemical and was used without further purification.

N-Succinimidyl pentafluorobenzoate was synthesized by the dropwise addition of pentafluorobenzoyl chloride (Aldrich, 2.2 ml, 15.3 mmol) to a stirred solution of N-hydroxysuccinimide (1.936 g, 16.8 -01) in DMF (12

ml) and diisopropylethylamine (2.6 ml, 15 mmol) under argon. The reaction mixture was stirred at room temperature for 2 h, poured into water (100 ml) and extracted with toluene (80 ml). The organic layer was washed with saturated NaHCO, (30 ml) and brine (30 ml), dried over MgS0, and the solvent was removed under reduced pressure. The brown solid was purified by column chromatography on a silica gel (Merck, 60-200 mesh) column (10 x 1 in i.d.), eluting with toluene (250 ml). The solvent was removed under reduced pressure to yield 3.50 g (74%) of a white solid: GC purity 94%, HPLC purity 85% (additional impurity most likely pentafluorobenzoic anhydride); MS, m/z (% abundance) 309 (Mf, 2), 195 (loo), 167 (62), 117 (51). N-Succinimidyl-4-carboxy-9-fluorenone was synthe-

sized by the addition of a solution of 9-fluorenone-4- carbonyl chloride (Aldrich, 3.60 g, 14.8 mmol) in DMF (25 ml) to a stirred solution of N-hydroxysuccinimide (1.91 g, 16.6 mmol) in DMF (15 ml) and diisopropylethylamine (2.6 ml, 14.9 mmol) under argon. The reaction mixture was stirred at room temperature for 1 h, poured into water (100 ml) and extracted with toluene (130 ml). The organic layer was washed with saturated NaHCO, (50 ml), which produced an orange precipitate (9-fluorenone-4-carboxylic acid), further washed with brine (50 ml), dried over MgSO, and the solvent was removed under reduced pressure. The orange solid was recrystallized from CHC1,-hexane to produce 2.16 g (45%) of an orange solid: GC purity 96%, HPLC purity 95%; MS, m/z (% abundance) 321 (M', l l) , 207 (loo), 179 (59), 151 (66), 150 (43).

N-Succinimidyl 3-nitrobenzoate was synthesized from the corresponding acid by standard methods.'j 3- Nitrobenzoic acid (Aldrich, 2.69 g, 16.1 mmol) and N- hydroxysuccinimide (1.86 g, 16.1 mmol) were dissolved in DMF (60 ml) under argon. Dicyclohexylcarbodiimide (3.31 g, 16.0 mmol) dissolved in DMF (40 ml) was added dropwise. The solution was stirred for 14 h at room temperature and filtered to remove the dicyclohexylu- rea. The reaction mixture was poured into water (150 ml) and extracted with toluene (200 ml). The organic layer was washed with brine (50 ml), dried over MgS04, and the solvent was removed under reduced pressure. Recrystallization from CHCl, yielded 2.59 g (61%) of a white solid: GC purity 97%, HPLC purity 95%; MS, m/z (% abundance) no M', 150 (100, 0,NC6H4CO), 104 (24, C,H,CO), 76 (25, C6H4).

Oligonucleotide synthesis

Oligonucleotides were synthesized on an Applied Bio- systems Model 392 DNA/RNA synthesizer using proto- cols and reagents for standard phosphoramidite chemistry from Applied Biosystems. The oligonucleotide used for the present work was the M13(-40) primer, 5'-GTTTTCCCAGTCACGAC-3' (isotope-averaged molecular weight 5121.4 Da), which has a mixed- base composition, a molecular weight in an easily accessible range for MALDI-MS experiments, and is used in sequencing reactions. The aminohexyl linker arm was incorporated at the 5'-terminus of the oligonucleotide in the final sequence on the automated DNA synthesizer by reaction with 6-(4-monometh-

ELECTROPHORE-LABELED OLIGONUCLEOTIDES 663

oxytrity1amino)hexyl- (2 - cyanoethyl) - (N,N - diisopro- py1)phosphoramidite (Glen Research) in CH3CN. “De- protection” was carried out in ammonia solution at room temperature (1 h) followed by heating at 40 “C (18 h). The MMT-protected oligonucleotide was purified with an Oligo-Pak column (MilliGen/Bioresearch Divi- sion of Millipore) following the recommended proto- cols, and the purity was confirmed by reversed-phase HPLC as described above. The MMT group was removed by treatment with 80% acetic acid. The modified oligonucleotide (see Fig. 1) was purified by ethanol precipitation. The unmodified M13( -40) primer was also synthesized for MALDI-MS sensitivity comparison and mass calibration.

Oligonucleotide derivatization

The synthetic route used to attach the electrophore to the linker arm of the oligonucleotide was similar to that used for the attachment of fluorescent labels to synthetic primers for fluorescence detection on sequencing gel^.'^-'^ The general procedure for derivatization is as follows: an aqueous solution of the aminohexyl- modified oligonucleotide (1.5 mM, 25 p1, 37.5 nmol) was added to a sodium borate buffer (0.05 M, pH 9.3, 75 pl) in a disposable plastic reaction tube. The desired electrophore derivative (250-fold excess) was added in 25-50 1-11 of DMF and the solution was mixed with a vortex mixer and stored in the dark at room temperature for 14 h. Although the electrophores are not very soluble in the reaction mixture and typically precipitate, the derivatization reaction occurs in high yields (see below). The mixture was diluted with water (100 pl), applied to a Pharmacia Sephadex (G-25M) PD-10 gel filtration column (50 x 15 mm i.d.), eluting with water, and 1 ml fractions were collected in order to remove excess electrophore. The fractions were analyzed by HPLC with the desired product eluted in the column void volume (fractions 2-5). The HPLC retention times of the products using the conditions outlined above are given in Table 1. Relative to the desired product, less than 6% of the unlabeled amino-modified oligonucleotide was typically observed by HPLC. The fractions were concentrated in a SpeedVac (Savant) and addi-

1 2 3 4

Figure 1. (A) Reaction of the aminohexyl modified 17-mer (5- GlllTCCCAGTCACGAC-3) with various electrophores (E). (B) Structures of the electrophores in the form in which they are attached to the oligonucleotide.

Table 1. Reversed-phase HPLC retention times for electrophore- labeled oligonucleotides

Retention time’ Sample’ (min)

17-mer 16.9 1 7-mer-C6NH, 16.6 17-mer-C,NH-1 21.9 17-mer-C,NH-2 20.2

17-mer-C,NH-4 19.9 17-mer-C,NH-3 20.4

a See Fig. 1 for electrophore structures. Hamilton PRP-1 column (250 x 4.1 mm

i.d.) with 0.1 M tri-ethylammonium acetate-CH,CN (95:5) and a linear gradient of CHJN from 0 to 75% over 40 min at 1 ml min-’.

tional purification of the product was accomplished by either a second gel filtration column, eluting with 0.1 M ammonium acetate, or reversed-phase HPLC as described above. Purities of the electrophore-labeled oligonucleotides were >95% by HPLC. The number of moles of isolated product was calculated from Beer’s law, using a calculated millimolar absorptivity for the chemically modified DNA at 260 nm of 152.2 1 mmol-’

tion of the electrophore to the absorbance at 260 nm is small (typically < 10%) based on the absorption spectra and the molar absorptivity of the products from the reaction of the electrophores with methylamine (as a model for the arylamide chromophore). The isolated yields were calculated from the number of moles of isolated electrophore-labeled DNA (by UV measurement) relative to the initial number of moles of DNA used in the synthesis. The isolated yields of the electrophore- labeled 17-mers were p-nitrobenzene (1) 11%, pentafluo- robenzene (2) 53%, 9-fluorenone (3) 73% and 3-nitrobenzene (4) 74%. Although the reactions are nearly quantitative by HPLC, the isolated sample yields are significantly less as a consequence of sample losses during purification. After purification, the samples were concentrated to dryness and stored at -20°C until further use. For MALDI experiments, the frozen samples were dissolved in deionized water.

cm-l , 2s and the volume of the sample. The contribu-

MALDI sample preparation

Typically, 1 pl of a 0.3 M solution of the matrix compound 3-hydroxypicolinic acidz6 (HPA, Aldrich) in 1 : 1 CH,CN-H,O) was mixed with 1 pl of a 1-100 pM solution of the labeled oligonucleotide, and 1 pl of the mixture was spotted on to a stainless-steel probe tip. To reduce the effects of alkali metal adduction, several NH,+-treated Dowex 50W-X12 cation-exchange beads (100-200 mesh, Bio-Rad) were also added to the sample.” The solvent was allowed to evaporate, leaving a spot containing small matrix crystals doped with DNA. The MALDI process only consumes a tiny

664 P. F. BRITT, G. B. HURST AND M. V. BUCHANAN

amount of the material contained in the sample spot, so the amount of oligonucleotide detected is much smaller than that actually applied to the probe tip.

MALDI mass spectrometry

The in-house-constructed time-of-flight mass spectrometer used for MALDI-MS experiments is shown sche- matically in Fig. 2. The probe tip bearing the dried sample spot is inserted into the vacuum chamber of the mass spectrometer. A pulsed nitrogen laser (Laser Pho- tonics LN203C, pulse width 600 ps, wavelength 337 nm) is fired at the sample, causing desorption and ionization. The laser is attenuated using a stack of glass microscope slides. A two-stage accelerating region supplies up to f 1 7 kV total potential to propel the resulting ions into a field-free drift region. To increase the efficiency of ion transport across the drift region, and to reduce the effects of abundant low-mass matrix ions, a bipolar pulsed electrostatic ion guide is used.28 The signal from the dual microchannel plate detector (Comstock CP-625-5O/S) is amplified (Ortec 9305 preamplifier) and recorded using a LeCroy 7200A digital oscilloscope. Data are subsequently transferred to a personal computer for analysis.

RESULTS AND DISCUSSION

The purpose of modifying oligonucleotides with an electrophore is to explore electron capture as a possible alternative to the normal proton transfer ionization mechanism observed in MALDI. Electron capture ionization should increase the yield of oligonucleotide anions by both increasing the ionization efficiency and

sample K Y n,

reducing fragmentation. Therefore, the electrophores selected for this study should have a high electron afin- ity and should undergo electron capture to form stable radical anions, M- ', without fragmentation. Two classes of compounds that fit these criteria are aromatic hydrocarbons with electron-withdrawing substituents, such as nitro or cyano groups, and polycyclic aromatic ketone^.^',^ These electrophores can be covalently bound to the oligonucleotide by coupling an aminohexyl-modified oligonucleotide (see Experimental) with an electrophore containing an amine reactive func- tional group, e.g. ihe N-succinimidyl ester of a carboxylic acid or an isothiocyanate derivative. Figure 1(B) presents the structures of the electrophores used in this study, shown in the form in which they are attached to the modified oligonucleotide by the primary aliphatic amine, i.e. through an amide or thiourea linkage.

The labeling strategy used in this study allows for the rapid screening of a wide variety of electrophores. The amine reactive electrophore derivatives can be easily prepared from commercially available starting materials and the labeling reaction can be conveniently followed by reversed-phase HPLC since the electrophore-labeled product has a longer retention time than the unmodified oligonucleotide, as shown in Table 1. One advan- tage of using a diode-array detector for HPLC analysis is that the absorption spectrum for each chromatographic peak provides insight into the identity of the material. The oligonucleotide has I,,, = 260 nm, while the electrophore-labeled product should have absorption bands from both the oligonucleotide and the electrophore. As shown in Fig. 3 for the reaction of p-nitrophenyl isothiocyanate with the aminohexyl- modified oligonucleotide, the peak at 16.6 min is unre- acted aminohexyl oligonucleotide (A,,, = 260 nm), and the peak at 21.9 min has the expected absorption spectrum for the oligonucleotide plus the p-nitrophenyl thiourea chromophore, I,,, = 344 nm. After purification on a Sephadex column and reversed-phase

I Microchannel

v1 v2 I \ 1 -2000 v

attenuation

Nirogen Laser (337 nm,

loo CIJlpulse)

I

I 486 Computer

Figure 2. Schematic diagram of the MALDI time-of-flight mass spectrometer.

ELECTROPHORE-LABELED OLIGONUCLEOTIDES 665

0.20

m 0 C ; 0.10

9 0 M

0.0

0 10 20 Retention Time (minutes)

Figure 3. HPLC of the reaction mixture of the aminohexyl oligonucleotide and p-nitrophenyl isothiocyanate after a Sephadex column (lower trace), and UV-visible absorbance spectra of the HPLC peaks eluting at 16.6 and 21.9 rnin.

0.4 4

10 20 30 40 Retention Time (minutes)

Figure 4. HPLC of the products from the reactions of N-succinimidyl pentafluorobenzoate with the arninohexyl-modified (eluting at 20.3 min, dashed line) and unmodified oligonucleotide primer (at 16.8 min, solid line). Peaks at 24.9, 30.1 and 30.7 min are mobile phase impurities.

HPLC, MALDI-MS analysis of the latter peak confirmed that it was the electrophore-labeled oligonucleotide (see below).

To verify that the electrophore reacted only with the primary aliphatic amine at the 5'-terminus of the primer rather than with the amines on the nucleic bases, an unmodified M13( - 40) primer (5'-terminus contains OH) was subjected to the coupling reaction conditions. Figure 4 shows the HPLC traces for the reaction mixtures, after separation on a Sephadex column, of both the unmodified oligonucleotide (solid line) and the arninohexyl-modified oligonucleotide (dashed line) with N-succinimidyl pentafluorobenzoate. The unmodified 17-mer was recovered unchanged, with no reaction products detected by HPLC or UV-visible absorbance analysis, indicating that only the primary aliphatic amine at the 5'-terminus of the modified 17-mer reacts with the amine-reactive electrophores under the reaction conditions described above.

Figure 5 shows the MALDI-TOF spectra of 3:2 (mo1e:mole) mixtures of the 5'-OH primer and the pentafluorobenzoyl-labeled (see 2, Fig. 1) primer. The labeled and unlabeled species, which differ in mass by 372 Da, are clearly resolved in both the positive-and negative-ion spectra. A small amount of fragmentation, leading to peaks at lower m/z than the main peaks, and also adduction of alkali metal ions and matrix fragments, leading to shoulders to higher m/z, are observed (see below).33 Doubly charged peaks appear in both

0.02 7-

- ln = 0 > v

.- 50.01 m c (u c c -

0

- 0.1 ln

0 > 1.

e - v

c ._ 0.05

c c -

I' 17-mer

2000 3000 4000 5000 6000 7000 mlz

Figure 5. MALDI mass spectra of unlabeled and pentafluorobenzoyl-labeled M13( -40) primer (2). For the negative-ion spectrum, the total accelerating potential was -1 3 kV and the sample loading was 5.1 pmol of unlabeled primer, 3.3 pmol of electrophore-labeled primer and 0.15 pmol of HPA. For the positive-ion spectrum, the total accelerating potential was +13 kV and the sample loading was 3.4 pmol of unlabeled primer, 2.2 prnol of labeled primer and 0.1 pmol of HPA. The field-free region was 1.5 rn in length for both spectra.


positive-and negative-ion spectra for the labeled and unlabeled species. Mass calibration for the MALDI- TOF spectra was carried out by assigning m/z values to peaks corresponding to the 5‘-OH primer and the doubly charged 5’-OH primer to calculate constants A and B in the expression m/z1I2 = At + B, where t is the flight time.34 The resulting calibration was used to esti- mate the m/z corresponding to the peak due to the labeled primer. Results similar to those shown in Fig. 5 were obtained for mixtures of the 5’-OH primer with primers labeled with the other electrophores studied (structures 1, 3 and 4 in Fig. 1). Table 2 lists measured and calculated molecular weights for the primers labeled with the different electrophores 1-4. The observed molecular weights of the electrophore-labeled compounds for 2-4 agree fairly well (within 0.1%) with the calculated molecular weights. Agreement for the 1- labeled primer is slightly worse (0.5%).

To determine the extent of fragmentation of the labeled primers within the MALDI-TOF accelerating region, MALDI mass spectra of the different labeled primers alone were obtained, and are shown in Fig. 6. Each spectrum shows a major peak corresponding to a product resulting from attachment of a single electrophore label at the 5’-terminus of the aminohexyl- modified primer. Significantly, little or no fragmentation resulting in loss of the electrophore labels is observed in the spectra. Loss of the electrophore plus the entire aminohexyl linker would yield peaks with m/z approx- imately that of the unlabeled primer, indicated by the vertical dashed line in Fig. 6. A minor peak corresponding to loss of roughly 102 mass units from the 9- fluorenone derivative 3 [Fig. 6(a)] is observed, but this fragment does not correspond to loss of the 9- fluorenone group itself, which would lead to a loss of 179 Da. It probably corresponds instead to the loss of a cytosine base, 110 Da, from the primer. The m-nitro- labeled primer 4 [Fig. 6(b)] also shows a small peak that corresponds to the loss of -129 Da, which could correspond to loss of a thymine base (125 Da) or, more likely, loss of the nitrophenyl group of the electrophore (122 Da). In the spectrum of the p-nitro-labeled primer 1 [Fig. 6(c)], a peak of unknown origin corresponding to loss of -160 Da from the intact labeled primer is observed. While these minor peaks in Fig. 6(a-c) could be due to prompt fragmentation, they could also be due

Table 2. Calculated molecular weights and observed m/z values for electrophortderivatized M13 (-40) primer, 5’-GT’ITTCCCAGTCACGAC-3’ ’

Calculated Observed m/f Observed m / 2 Compound MWb (positive ions) (negative ions)

1 -Derivatized primer 5480.8 5457 5468

3-Derivatized primer 5506.8 5507 5508 4-Derivatized primer 5449.7 5443 5442

a Calculated isotope-averaged molecular weight of underivatized primer = 51 21.4 Da.

Calculated isotope-averaged mass for the neutral molecule, assuming that all phosphates are paired with protons.

Observed values obtained from a two-point internal calibration using the singly and doubly charged 5-OH primer.

2-Derivatized primer 5494.7 5498 5494

1lmv a

I I I I1

L I ‘ . . I , T ’OmV I b I I I 1 1 I 1

I I I C

I

! A U d . II I II

10 mV d

2000 3000 4000 5000 6000 7000 m/z

Figure 6. MALDI negative-ion mass spectra of electrophore- labeled M13(-40) primers. The dashed vertical line is at the m/z of the underivatized (i.e. 5-OH) primer. (a) 9-Fluorenone derivative (3), -1 6 kV total accelerating potential, 10 pmol on probe tip. (b) m-Nitrobenzene derivative (4). -1 6 kV total accelerating potential, 10 pmol on probe tip. (c) p-Nitrobenzene derivative (I) , -13 kV total accelerating potential, 2 pmol on probe tip. (d) Pentafluoro- benzoyl derivative (2). -17 kV total accelerating potential, 7 pmol on probe tip. For spectra (a), (b) and (d), the field-free drift region was 2 m in length, a sampling interval of 5 ns was used and 0.3 pnol of HPA matrix was used. For spectrum (c), the field-free drift region was 1.5 rn in length, a sampling interval of 50 ns was used and 0.1 5 pmol of HPA matrix was used.

667 ELECTROPHORE-LABELED OLIGONUCLEOTIDES

to degradation of the samples, which were stored as unbuffered solutions. In contrast to the other three derivatives, the pentafluorobenzoyl derivative 2 [Fig. 6(d)J shows no evidence of either fragmentation or degradation, despite freezer storage in unbuffered solution for more than 1 year. The spectra in Fig. 6 indicate that the electrophore-labeled primers can be successfully synthesized, that the labels for the most part remain attached to the primer throughout the sample preparation and laser desorption steps of the MALDI-MS measurement process and that sensitivity in the picomo- le range can be obtained.

Conventional negative-ion mass spectra of small molecules that have strong electron-capturing character- istics are generally obtained by negative-ion chemical ionization, in which a high pressure of a buffer gas is present in the mass spectrometer source to provide multiple collision conditions for electrons, thus slowing them to near thermal velocities to allow intimate contact with the analyte molecules." Our initial hypothesis was that the plume produced by the laser desorption event in MALDI might contain a local high- pressure region that could offer similar conditions, leading to electron capture by electrophore-labeled analytes in the MALDI plume. The results in Fig. 5 suggest that conditions for efficient electron attachment by the electrophore-derivatized primer are not present in normal MALDI experiments. If efficient electron attachment were occurring, two effects would be expected. First, the signal due to the labeled primer would be more intense than that due to the unlabeled primer in the negative-ion spectrum. Second, the ratio of signals due to the labeled and unlabeled primers should differ in positive- and negative-ion spectra. Neither of these effects has yet been observed. The reason is probably that conditions conducive to electron capture (high pressure, multiple collisions) are not present in the MALDI-TOF instrument. Therefore, we are currently exploring different strategies for modifying the mass spectrometric measurement to provide more

favorable conditions for electron capture by the labeled oligonucleotides. While a MALDI source at elevated pressure (similar to that used for negative-ion chemical ionization) might be interfaced with the TOF, losses due to differential pumping requirements might negate any sensitivity gain afforded by electron capture ionization. Delayed acceleration for MALDI-TOF, in which the source region containing the sample is field-free during a delay following the laser pulse,35 offers new pos- sibilities for promoting electron capture conditions. Pre- liminary delayed extraction MALDI-TOF experiments on a 2-labeled DNA tetramer yielded isotopically resolved peaks (data not shown). Under various MALDI conditions, comparison of the observed inten- sity pattern of these peaks with the calculated isotope distributions for the [M - HI- and M-' species should provide more detailed information about relative con- tributions of the two ionization mechanisms (proton abstraction versus electron capture).

Because of the promising results obtained for synthesis and robustness under MALDI conditions of electrophore-labeled oligonucleotides, we plan to pursue instrumental modifications that will favor electron capture ionization. One possibility is, in com- bination with delayed extraction, to flood the TOF source regon with low-energy electrons from an electron monochr~mato r~~ during the delay time following the laser pulse when the source region is field-free.

Acknowledgement

We thank Robert Foote (ORNL Biology Division) for the synthesis of the 17-mers and Fred Sloop, Larry Waters, Robert Hettich (ORNL Chemical and Analytical Sciences Dwision) and Elizabeth Stemmler (Bowdoin College) for helpful discussions. This research was spon- sored by the National Institutes of Health, National Center for Human Genome Research, Grant No. 1 R55 HG/OW0819-01Al, under Interagency Agreement 1884-FO26-Al with the US Department of Energy under Contract DE-AC05-960R22446 with Oak Ridge National Laboratory, managed by Lockheed Martin Energy Research colp.

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synthesis of electrophore-labeled oligonucleotides and characterization by matrix-assisted laser...

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