Rapid extraction and structural characterization of biomolecules in

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Anal. Chem. 1993, 65, 1329-1335

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Rapid Extraction and Structural Characterization of Biomolecules in Agarose Gels by Laser Desorption Fourier Transform Mass Spectrometry Jocelyn C. Dunphy$J K. L. Busch$ R. L. Hettich,’*sand M. V. Buchanad School of Chemistry and Biochemistry, Georgia Znstitute of Technology, Atlanta, Georgia 30332-0400, and Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830-6120

A method originally developed for the extraction of biomolecules from agarose gel slices has been utilized as a rapid means of isolating biological compounds from gels for subsequent structural characterizationby matrix-assistedlaser desorption-ionization Fourier transform mass spectrometry (MALDVFTMS). This “freeze-squeeze” extraction method involves pressure extrusion of fluid from frozen gel slices and provides near 50% recovery of analyte in less than 5 min. Experiments were directed at examining the recovery efficiency of the extraction method using 14Clabeled adenosine monophosphate and investigating the effect of high buffer concentrationson the laser desorption mass spectra. When coupled with this extraction technique, MALDVFTMS can be used to detect and identify biomoleculesat the low picomole level in agarose gel slices. The accurate mass measurements and MS/MS capabilities of the FTMS were exploited to provide detailed structural information at the isomeric level for oligonucleotideselectrophoresed into agarosegels.

INTRODUCTION Slab gel electrophoresisis a powerful method for separating and purifying biological molecules such as nucleic acids and proteins.lJ T w o types of gel media, agarose and polyacrylamide, are commonly used today. Agarose is a linear galactan polymer, while polyacrylamide is a polymerization product of acrylamide monomer and a cross-linking comonomer, usually N’JV-methylenebisacrylamide. The gel medium employed for a given separationis dictated largely by tradition, because either medium can be modified to suit a given application. Considerations of the size range of the biomolecules to be separated, the spatial resolution required, and the subsequent manipulation that is to be performed with the separated biomolecules influence the choice of the gel. In general, agarose gels with their larger pore size are used for the separation of the largest biological molecules, i.e., large proteins and large DNA fragments. Polyacrylamide gels are used for smaller proteins and smaller DNA fragments (100 000 Da or less). However, newer grades of agarose gels are being

* Author to whom correspondence

should be addressed. Georgia Institute of Technology. f Present address: Proctor and Gamble Co., Cincinnati, OH 45241. i Oak Ridge National Laboratory. (1)General review of nucleicacid electrophoresis: Gel Electrophoresis of Nucleic Acids: A Practical Approach; Rickwood, D., Hames, B. D., Eds.; IRL Press, Ltd.: Oxford, U.K., 1982. (2) Discussion of peptide and protein electrophoresis: Gel Electrophoresis and Isoelectric Focusing of Proteins; Allen, R. C., Saravia, C. A., Maurer, H. R., Eds.; Walter de Gruyter Press: New York, 1984. t

0003-2700/93/0365-1329$04.00/0

developed to allow resolution of the smaller biomolecules. Agarose gels do provide a few practical advantages over polyacrylamide gels. Agarose is nontoxic, while acrylamide monomer is an accumulative neurotoxin. Because agarose is gelled without the aid of a catalyst (the packaged agarose powder is simply dissolved in aqueous buffer and poured), it allows quick and easy staining and can readily be dried.2 Methods of detectingthe separated bands in electrophoretic slab gels have traditionally involved either staining with dyes (such as Coomassie Blue, Amido Black, and Ponceau S), preelectrophoretic radiolabeling followed by postelectrophoretic autoradiography,or complexationwith a fluorescent stain such as ethidium bromide. While such detection methods can be sensitive, allowing the detection of subnanomoles of analyte per band, they do not provide any structural information for the biomolecules. In addition, the handling and disposal of radiolabeled compounds make this method more difficult. The ideal detector for gel electrophoresis would provide high sensitivity detection as well as accurate molecular weight and structural information for unknown bands that may be criticalto the biochemist. Recent advances in mass spectrometric (MS) techniques, such as matrix-assisted laser desorption-ionization (MALD1)- and electrospray ionization,&”Jindicate that mass spectrometry can be used for the soft ionization of biomolecules with molecular masses greater than 100 OOO Da. These developments, combined with the capabilities of mass spectrometry for sensitivedetection and detailed structural characterization of unknown compounds, indicate that mais spectrometry should be well-suited for the examination of biological compounds separated by gel electrophoresis. Combinations of electrophoretic separations with mass spectrometry have only recently been reported. The advent of capillary zone electrophoresis (CZE) has led researchers to develop CZE/MS interfaces based on electrospray ionization11-13and fast-atom bombardment (FAB)ionization.l4J5 These continuous-introduction ionization techniques can easily be used with CZE because this form of electrophoresis is conducted in a stream of buffer solution in a capillarytube. The predominant challenge in performing mass spectral (3) Karaa, M.; Hillenkamp, F. Anal. Chem. 1988,60,2299. (4) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.;Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 251. (5) Beavie, R. C.; Chait, B. T. Rapid Commun. Mass Spectrom. 1989, 3, 233. (6) Whitehouse, C. M.; Dreyer, R. N.; Yamaahita, M.; Fenn, J. B. Anal. Chem. 1985,57, 675. (7) Fenn, J. B.; Mann, M.; Meng, C. K. Science 1989,246, 64. (8)Mann, M.; Meng, C. K.; Fenn, J. B. Anal. Chem. 1989,61,1702. (9) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Science 1990,248,201.

(10)Smith, R. D.; Barinaga, C. J. Rapid Commun. Mass Spectrom.

1990. - _ _r_4. 54. -I

(11) Smith, R. D.; Olivareg J. A.; Nguyen, N. T.; Udseth, H. R. Anal.

Chem. 1988,60, 436. (12) Udseth, H. R.; Loo, J. R.; Smith, R. D. Anal. Chem. 1989,61,228. (13) Lee, E. D.; Mueck, J. D.; Henion, J. D.; Covey, T. R. Biomed. Enuiron. Mass Spectrom. 1989,18, 253. @ 1993 Amerlcan Chemical Society

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analysis of gel electropherograms is the detection of sample molecules that are inaccessible when contained in a fully hydrated, complex gel network. In biochemical laboratories, recovery or isolation of analytes from gels is often required for subsequent chemical or enzymatic procedures that may be required. Common methods of sample isolation include extended solvent extractions or grinding,1621capillary blotting onto nitrocellulose membrane (Southern b l ~ t ) , ~or*elec$~~ troblotting (Western blot).24,*5More recently, peptides in agarose gels have been analyzed by disruption of the gel on a microscale with a piezoelectric homogenizer and mass spectral introduction of the resulting liquid via a continuousflow FAB probe.26 While FAB-MS has proved very useful in analyzing compounds in gel electropherograms, its disadvantages for this application have included an upper mass limit of approximately 10 000 Da, a large amount of “chemical noise” from the FAB matrix, and poor detection limits. In most of the work reported above, several micrograms of material were present in the gel bands examined by mass spectrometry. The FAB ionization method may not have been the only limiting factor in the sensitivity in the above-mentionedwork, as it is likely that low recovery yields of the compounds from the gels may also have been a major limitation. Recently developed matrix-assisted laser desorption techniques have improved the detection limits and application of mass spectrometry for biological molecules. With these techniques, mass spectra have been obtained from picomoles of peptides over 100 000 Da in molecular m a ~ s . ~Without -~ the matrix present, only low-mass fragment ions from these large biomolecules would be observed under normal LD conditions. MALDI has been mainly used with time-of-flight (TOF) mass analyzers, which provide a mass range exceeding 300 000 Da. The resulting mass spectra usually reveal only the molecular ions, with no fragmentation indicative of the ion structures. For example, matrix-assisted TOF has been used to examine small oligonucleotides and reveals (M + H)+ ions with virtually no fragmentation.*: Matrix-assisted laser desorption is also useful for enhancing the detection and upper mass limit for analysis of peptides and oligonucleotides with Fourier transform mass spectrometry (FTMS) as ell.^^^^^ Recent research has indicated that careful control of iontrapping parameters enables the mass range of MALDI/ (14) Moseley, M. A.; Deterding, L. J.; Tomer, K. B.; Jorgenson, J. J . Chromatogr. 1989, 480, 197. (15) Caprioli, R.; Moore, W.; Martin, M.; DaGue,B.; Wilson, K.; Moring, S. J . Chromatogr. 1989, 480, 247. (16) General review of recovery methods: Smith, H. 0. Methods Enzymol. 1980, 65, 371. (17) Maxam, A. M.; Gilbert, W.Proc. Natl. Acad. SCL.U.S.A. 1977,74, 560. (18)De Wachter, R.; Fiers, W. Anal. Biochem. 1972, 49, 184. (19) Wheeler, F. C.; Fishel, R. A.; Warner, R. C. Anal. Biochem. 1977, 78, 260. (20) Bustin, M.; Hopkins, R. B.; Isenberg, I. J . Biol. Chern. 1978, 253, 1694. (21) Pedersen, F. S.; Haseltine, W. A. Methods Enrymol. 1980, 65, 680. (22) Southern, E. M. J . Mol. Biol. 1975, 98, 503. (23) Bowen, B.; Steinberg, J.; Laemmli, U. K.; Weintraub, H. Nucleic Acids Res. 1980, 8, 1. (24) Towbin, H.;Stahelin,T.;Gordon,J.Proc.,~at~.Acad.Sci. U.S.A. 1979, 76, 4350. (25) Burnett, W. N. Anal. Biochem. 1981, 118, 195. (26)Brown, S. M.; Busch, K. L. Proceedings of the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tucson, AZ, June 3-8, 1990; p 1325. (27) Spengler, B.; Pan, Y.; Cotter, R. J.; Kan, L . 3 . Rapid Commun. Mass Spectrom. 1990, 4, 99. (28) Hettich, R. L.; Buchanan, M. V. J . Am. SOC.Mass Spectrom. 1991,2, 22. (29) Hettich, R. L.; Buchanan, M. V. J . Am. SOC.Mass Spectrom. 1991, 2, 402.

FTMS to be extended beyond miz 20 OO0.30 Further research is in progress to refine the matrix-assisted technique for FTMS while utilizing the ion manipulation and structural characterization capabilities that have already been developed for this technique. This paper focuses on the development of an interface between agarose gel electrophoresis and MALDI/ FTMS that minimizes the sample preparation time required and exploits the low detection limits and ion structural characterization capabilities of the FTMS technique. Although the results of the “freeze-squeeze” extraction are demonstrated with small oligonucleotides, this extraction technique should be useful for larger biomolecules as well. Continued development of methods to increase the mass range of MALDI/FTMS for large biomolecules should enhance the general application of this freeze-squeeze method. EXPERIMENTAL SECTION Samples and Reagents. The oligonucleotide dimers and tetramer were obtained either from Sigma Chemical Co. or Pharmacia LKB Biotechnology,while the oligonucleotidetrimer was donated by Dr. Robert Foote of ORNL’s Biology Division. All of these compounds were used without further purification. The LD matrix compound, 2-pyrazinecarboxylic acid, was used as obtained from Aldrich Chemical Co. Introduction of Analyte into Gel Slices. For preliminary studies, gel samples were prepared by soaking small slices of low-melting preformed agarose gels (donated by Dr. Dorothy Skinner of ORNL’s Biology Division) in solutions (1-mL total volume) of analyte in varying concentrations for several hours. The gel slices used were approximately the size that would contain a single analyte band followingelectrophoresis (5 x 5 x 0.1 mm) and, when fully hydrated,were capable of retaining approximately 50 pL of buffer solution. After being soaked in the analyte solution, the gels were rinsed in distilled water prior to analysis to remove traces of the analyte from the gel surface. For later experiments, samples were prepared by introducing analyte into the gel slices via electrophoresis. In this method, the gel slice was mounted on a grounded platinum foil, and a Teflon ring was positioned on top of the gel slice, the center opening of which formed a seal on the gel surface. Analyte buffer solution (25 pL, pH 8.1) was added to the opening, allowing electrical contact with the top of the gel slice. A platinum wire (32 gauge) was immersed into the solution, and a potential was applied (usually around negative 50-80 V for 1-5 min) to induce electromigration of the charged analyte molecules into the gel toward the grounded electrode. For these experiments, the agarose gel (3%) was made from NuSieve agarose (FMC Corp.) buffer (pH &l), which in a 8.2 mM tris-phosphate-EDTA (TPE) was the same buffer used to dissolve the analytes. Individual excised gel slices were 5 X 5 x 2 mm in size and contained approximately 100 pL of fluid when hydrated. Preparation of Gel Slices for LDiFTMS Analysis. Two methods of preparing the agarose gel slices for mass spectral analysis were examined. The first method involved the direct analysis of the analyte-doped gel. This approach required attaching the gel slice to the tip of the laser probe and allowing the vacuum in the sample lock to dry the gel, shrinking it in size. After dehydration in this manner, 10pL of the LD matrix solution (approximately 120mM aqueous solution of 2-pyrazinecarboxylic acid) was added to the dried gel slice, which was then analyzed directly by LDIFTMS. The second sample introduction technique is a modification of the freeze-squeeze technique, originally proposed as a means of rapid recovery of long DNA from agarose Using this method, the analyte-doped gel slice was frozen for a few seconds in liquid nitrogen and then manually squeezed between two pieces of Parafilm, onto which most of the interstitial fluid containing the analyte was extruded. According to the original researchers.31approximately 70-80 3’ % of the gel weight (30) Castoro, J. A.; Koster, C.; Wilkins, C. Rapid Commun. Mass Spectrom. 1992, 6 , 239. (31) Thuring, R. W. J.; Sanders, J. P. M.; Borst, P. Anal. Biochem. 1975, 66, 213. (32) Wieslander, Id. Anal. Biochem. 1979, 98, 305

ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993

can be recovered as fluid in this manner. The fluid drop on the Parafilm was transferred to a stainless steel disk, and the matrix solution was directly added to the analyte drop on the disk. Recovery Study. The sample recovery achievable with the freeze-squeeze method was determined by performing the complete sample preparation and isolation procedure with a radiolabeled [8-14C]adenosine5’-monophosphate obtained from ICN Radiochemicals, Inc. This radiolabeled compound was supplied by the manufacturer as a 155 ng/pL solution in 20% ethanol. An aliquot of this solution was diluted with 8 mM trisphosphate-EDTA buffer to a final concentrationof 15.5 ng/pL. Liquid scintillation counting was used to compare standard solutions of this compound with solutions that had been spiked into and recovered from gels in order to quantitate the recovery of biomolecules from the gels. Each sample was counted for a total of 4 min with a Packard C-2425Tri-Carbliquid scintillation counter. Mass Spectrometry. Laser desorption-ionization was accomplishedby focusingthe fourth harmonic (266nm) of a QuantaRay DCR-11 Nd:YAG pulsed laser to approximately 0.2 mm in diameter onto the stainless steel sample target of the direct insertion probe, providing laser power densities in the 106-107 W/cm2 region. Ions generated by a single laser pulse were manipulatedand detected in the source cell of the Extrel FTMS2000 i n ~ t r u m e n t .For ~ ~ most of the mass spectra shown here, signal averagingof five to six individual laser shots was performed while the probe was rotated to expose fresh sample surface. The pressure in the source cell during excitation and detection was typically 1 X 10-7 Torr. The time domain signal was sampled under medium resolution conditions (32-128K data points) to obtain the mass spectra shown below. In many cases, low-mass interfering ions, arising primarily from the matrix, were ejected from the cell with a broad-band radiofrequency sweep prior to excitation and detection. For MS/MS experiments,the parent ion of interest was isolated in the FTMS cell by ejecting all other ions. The parent ion was then accelerated into a static pressure Torr, to induce collisions of argon, at approximately 5.0 X and subsequentdissociation. Collisionenergiesranged from 100 to 500 eV in the laboratory frame of reference.

RESULTS A N D D I S C U S S I O N Laser desorption can be used to examine compounds directly in a gel matrix. As described in the Experimental Section, a small piece of agarose gel containing a biomolecule such as a nucleoside was dried in the vacuum chamber and then rehydrated with the laser desorption matrix solution. This final mixture was then inserted into the mass spectrometer for examination by laser desorption. Direct laser desorption of this analyte-doped gel generated ions which identified the biomolecule present in the gel. However, because the analyte is entrained in the gel network and not simply adsorbed on the surface, the detection limits for direct laser desorption were determined to be quite poor. In addition, the gel matrix dramatically affects desorption and subsequent ionization of the biomoleculeas well as producing background ions in the mass spectra from the gel itself. It was found that the freeze-squeeze method of recovery improved the detection limits by at least an order of magnitude over those obtained with the direct desorption method as well as diminishing the background ions from the gel. Recovery Study with [14C]Adenosine 5’-Monophosphate (AMP). Because gel electrophoresis is usually performed on nanogram to microgram quantities of material, recovery efficiencies of analyte from the gels and instrumental detection limits must be compatible with these sample amounts. To estimate the recovery available with the freezesqueeze procedure, the following experiment was performed. A 25-pL aliquot of the 15.5ngfpL solution of [8-l4C1adenosine (33)For further details on the capabilities of FTMS, see: Fourier Transform Mass Spectrometry; Euolution, Innovations, and Applications, Buchanan, M. V., Ed.;ACS Symposium Series 359;American Chemical Society: Washington, DC, 1987.

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Table I. Results of the Freeze-Squeeze Recovery Study Using [ WIAdenosine 5’-Mono~hos~hate vol

total counts

amt (na)

25 10 65

471944 12 166 211548

381.5 9.8 176.4

(ALL) starting sample solution “used”sample solution recovered gel fluid

anal&-

% electromigated into gel 1 - 12166/477944X 100% = 98% % recovered 217548/(477944- 12166)X 100% = 47%

5’-monophosphate was analyzed by liquid scintillation counting (4 min), yielding 477 944 counts, as shown in Table I. Another 25-pL aliquot of the 15.5 ng/pL solution was loaded into the electromigration apparatus, as described previously. A negative potential (-40 V) was applied to this solution for 5 min to electrodeposit the sample into a hydrated agarose gel slice (containing approximately 50 pL of buffer solution). After this procedure, 10 pL of solution was recovered from the electromigration apparatus (the other 15 pL had been absorbed by the gel) and analyzed to determine the amount of sample that was excluded from the gel. The results in Table I indicate that 98% of the [%]AMP was deposited into the agarose gel by electrophoresis. Freeze-squeeze extraction of the doped gel yielded 65 pL of solution, which was then examined by liquid scintillation counting. The total counts indicated that 47 % of the [l4C1AMPcould be recovered from the gel. Although the original developers of the freezesqueeze extraction method predicted that 70-80% of the gel weight can be extruded as fluid,31%*this value does not necessarily indicate the recovery efficiency of analyte from the gel. The recovery efficiency of approximately 50% as determined above is encouraging, especially in light of the fact that this recovery should not be dependent on the molecular size of the analyte contained in the gel. Further refinements in the freeze-squeeze technique may improve the sample recovery. This recovery efficiency is not meant to be interpreted as a rigorous value for the extraction efficiency of all biomolecules from agarose gels, but is rather a general estimate of the quantitative recovery efficiencies which could be expected for extracting biomolecules from agarose gels. A different experiment with [l4C1AMPwas performed to assess the consumption of sample by the matrix-assisted LD experiment. A 15-ng sample of the labeled compound was deposited with 10 pL of 2-pyrazinecarboxylic acid onto the probe tip, and mass spectra were acquired from 30 laser shots on the deposited sample. Liquid scintillation counting of the remaining material on the probe was compared with another 15-ng aliquot of the standard [14C]AMP solution. Within experimental error, the amount of sample consumed in the laser desorption-ionization process could not be detected (most likely in the attomole to femtomole range). This experiment verifies that sample handling (gel loading and extraction), rather than instrumental factors, currently controls the achievable detection limits in matrix-assisted LD/FTMS analysis. Examination of Oligonucleotides Electrophoresed into Agarose Gel Slices. The results of the experiments described above indicated that the freeze-squeeze method provides a rapid, sensitive method of extracting the analyte contained in gel slices for mass spectral analysis. In addition, analyte recoveries attainable indicated that the technique, when combinedwith LD/FTMS, should provide low detection limits (picomole or less). The following experiments were designed to determine how well the overall strategy would

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perform under realistic conditions of sample introduction into gels, using oligonucleotides as model compounds. The goals of these experiments were to characterize the sample preparation procedure with regard to the presence of ionic contaminants (i.e., buffers contained in the gel) and their effect on the mass spectral response, to determine the types of structural information available from matrix-assisted LD/ FTMS analysis of oligonucleotides in agarose gels, and to ascertain the lowest amount of analyte deposited into the gel that would yield detectable mass spectral signal for oligonucleotide dimers, trimers, and tetramers. Effect of Buffer Constituents. Initial experiments aimed at recovering analyte from gel slices hydrated with buffer solution indicated that there might be difficulties in obtaining mass spectralinformation due to matrix effects from the buffer constituents. For example, direct analysis of oligonucleotides dissolved in high concentrations (>80 mM tris-phosphateEDTA) of aqueous buffer commonly used to compose gels and perform electrophoresis did not reveal ions characteristic of the oligonucleotide. The difficulty seemed to arise from the fact that when the solution was evaporated on the stainless steel probe tip, a sticky, partially hydrated residue remained that prevented ion formation by laser desorption. The formation of the sticky residue could by avoided only by adding a great excess of matrix solution compared with the volume of buffer solution deposited. In this case, the matrix/ analyte ratio was so great that analyte ions were not observed in the mass spectra. Because the freeze-squeeze method requires near complete recovery of the gel fluid, the above difficulty needed to be resolved for practical mass spectrometric analysis. It was found that use of a dilute buffer (such as 5-15 mM tris) was still sufficient to perform the electrophoresis and increased the quantity of buffer solution that could be evaporated on the probe before the formation of the intractable residue. Application of the LD matrix solution to the probe tip after this buffer solution had been evaporated resulted in a relatively thin crystalline film that yielded analyte ions upon laser desorption. The ability of FTMS to eject background ions resulting from the buffer and matrix constituents improves the dynamic range of the experiment, allowing a matrix/analyte ratio of approximately 1OOOO:l (w/ w), while diminishing the effects of high relative concentrations of buffer. Beavis and Chait have reported the use of sinapinic acid (which has a strong UV absorption at 355 nm, the third harmonic of the Nd:YAG laser) as a matrix for LD of proteins.34 These researchers determined that ionic contaminants did not interfere with the ionization process enhanced by this matrix. Examination of Oligonucleotides. Three oligonucleotide dimers, 5'-d(TC)-3', 5'-d(CT)-3', and 5'-d(AA)-3', were examined with the technique. Solutions of these dinucleotides were made in 8.2 mM tris-phosphate-EDTA buffer a t 1000, 100,10,and 1.0 ng/pL concentrations. Aliquots (25 pL) were loaded into the electromigration apparatus, and the appropriate potentials were applied. Maximum analyte quantities of 25 pg, 2.5 pg, 250 ng, and 25 ng, respectively, were thus electrophoresed into individual agarose gel slices 5 x 5 X 2 mm in size. The gel slices were then subjected to the freezesqueeze procedure as described previously. On average, 50 pL of fluid (approximately 50% of the hydrated gel volume) was recovered for each gel slice. The mass spectra of oligonucleotide dimers and all subsequent oligonucleotides a t high sample levels (25 or 2.5 pg) were obtained with an ion ejection step that removed most low-mass ions. In these experiments, the ions a t m/z 123 and 124, arising from 2-pyrazinecarboxylic acid, do not dominate the mass spectrum. For lower sample levels, ions of m/z

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Flgure 2. C I D MSlMS spectra of m/z 530, the deprotonated molecule of (a) 5‘d(TC)3’ and (b) 5‘d(CT)-3‘. from samples recovered from agarose gel slices containing 2.5 pg of analyte.

3‘ terminus, appear a t mlz 306 for 5’-d(TC)-3’ and mlz 321 for 5’-d(CT)-3’, as has been reported previou~ly.~~ This fragmentation pattern was also observed in negative ion FAB studiess38 and PDMS studies39g4’J of oligonucleotidesas well. Sequence ions correspondingto phosphate ester cleavage from either direction are observed, although the predominant fragmentation pathway in both cases appears to be 5‘phosphate ester cleavage with charge retention on the 3’ terminus of the oligonucleotide.29 This preferential fragmentation pathway simplifies sequence deduction from the mass spectraof oligonucleotides. For eachof the dinucleotides in Figure 2, an ion correspondingto the deprotonated nucleic base from the 5’ end of the molecule is also observed. In the CID MS/MS spectrum of 5’-d(TC)-3,deprotonated thymine is observed a t mlz 125, and in the corresponding 5’-d(CT)-3’ CID MSIMS spectrum, deprotonated cytosine is seen at mlz 110. An additional ion is observed in the CID mass spectrum of 5’-d(CT)-3’a t mlz 195. This ion corresponds to loss of the nucleic base (thymine in this case) from the nucleotide and is observed for most oligonucleotides. While 2.5 pg of dinucleotides contained in the gel slices could easily be observed, mass spectra could also be obtained for gel slices containing, at maximum, 25 ng of dinucleotide. Figure 3 shows the mass spectra of the dinucleotide isomers extracted from gels a t this sample level. To obtain these mass spectra, a broad-band rf ejection pulse was applied to (36) Grotjahn, L.; Frank, R.; Bloecker, H. Znt. J. Mass Spectrom. Zon Phys. 1982,46,439. (37) Cerny,R. L.; Gross, M. L.; Grotjahn,L. Anal. Biochem. 1986,156, 424. (38) Cerny, R. L.; Tomer, K. B.; Gross, M. L.; Grotjahn, L. Anal. Biochem. 1987,165, 175. (39) McNeal, C. J.; Ogilvie,K. K.; Theriault, N. Y.; Nemer, M. J. J. Am. Chem. SOC.1982,104, 981. (40) Viari, A.; Ballini, J.-P.; Vigny, P.; Shire, D.; Dousset, P. Biomed. Enuiron. Mass Spectrom. 1987, 14, 83.

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m/z Flguro 3. Matrlxasslsted negathre ion LDlFTMS spectra of (a) 5‘d(TC)3’ and (b) 5‘d(CT)-3’, obtained from agarose gel slices contalning 25 ng of analyte. Methionine enkephalin, mlz 572, was added as an Internal cailbrant in (b). eject ions below mlz 450. Because signal for the deprotonated molecular ions is low in absolute abundance, some low-mass background ions are still observed. However, the deprotonated molecular ions are still easily detected at this sample level. To obtain the mass spectrum of 5’-d(TC)-3’, 15 pL of extruded fluid (approximately 15% of the original hydrated gel volume) was added to 15 p L of matrix solution, and for 5’-d(CT)-3’, 20 pL of extruded fluid (approximately 20% of the hydrated gel volume) was added to 10 pL of matrix solution. As stated above, considerations of extraction efficiencyand sampleloading indicatethat samplescontaining less than 5 ng (10 pmol) were actually applied to the probe for each spectrum. Note that the detection limita obtained for the freeze-squeeze extraction method are virtually identical to those obtained for pure nucleic acid constituenta,36 implying that the background extracted from the agarose gel does not severely affect analyte detection. In the mass spectrum of 5’-d(CT)-3’, Figure 3b, 100 ng of methionine enkephalin was added to the sample-matrix mixture to provide an additional ion for mass scalecalibration. The ion a t mlz 572 corresponds to the deprotonated molecular ion of methionine enkephalin. Using the calculated values of mlz 572.2185, the (M - HI-of methionine enkephalin, and the 2-pyrazinecarboxylicacid background ions at mlz 123.0200 and 124.0278 as internal mass calibranta, the ion at nominal mlz 530 was measured to be mlz 530.1289. This measured value is within 1millimass unit of the calculated value for the (M - H)- of 5’-d(CT)-3’, which is mlz 530.1294. Examination of d(AA)yielded similar detection limita with the freeze-squeeze LDIFTMS approach as did 5’-d(TC)-3‘ and 5’-d(CT)-3’. A negative ion mass spectrum could be obtained for d(AA) eluted from a gel slice containing, at maximum, 25 ng of analyte. The (M - H)- ion a t mlz 563 was prominent at this low sample level. These results indicate that the detection limits for extraction of dinucleotides from

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ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993

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Figure 4. Matrix-assisted LD/FTMS spectra of 5’d(TGT)-3‘, obtained with the freeze-squeeze method from an agarose gel slice containing (a) 25 and (b) 2.5 pg of anaiyte. agarose gels and subsequent examination by matrix-assisted LD/FTMS are in the low nanogram (low picomole) region. The freeze-squeeze LDiFTMS technique was extended to examine an oligonucleotide trimer, 5’-d(TGT)-3’. Gel slices containing varying amounts of 5’-d(TGT)-3’ were analyzed. Figure 4 shows the mass spectra obtained from analyzing gel slices containing 25 and 2.5 pg of sample. A 7-pL sample of recovered solution (approximately 7 % of the hydrated gel volume) was added to 10 pL of matrix solution to obtain the mass spectrum in Figure 4a, while 15 pL of gel fluid (15% of the hydrated gel volume) was added to 10pL of matrix solution for the mass spectrum in Figure 4b. To obtain both mass spectra, ions below m/z 450 were ejected prior to detection. Of the sample ions, the deprotonatedmolecular ion is observed at miz 874, and an abundant 3’ sequence ion is observed at m/z 650. These mass spectra are quite similar to the standard matrix-assisted LD mass spectrum of this compound.29 The oligonucleotide tetramer 5’-d(AGCT)-3’ was also examined in this study. For reference, the standard matrixassisted LD mass spectrum of this compound is shown in Figure 5a. Ions below m/z 300 were ejected to obtain this mass spectrum. An abundant deprotonated molecular ion at m/z 1172 was observed, as well as laser-induced 3’ sequence ions at miz 939, 610, and 321. The entire sequence of this tetranucleotide is thus determined directly from the singlestage mass spectrum,as observed for other tetranucle~tides.~~ The ion at m/z 739 results from loss of neutral thymidine nucleoside and of neutral cytosine base, with charge retention on the 5’ end of the molecule, as determined by accurate mass measurements. Analogous ions were observed in a highenergy MSiMS study of oligonucleotide trimer and tetramer ions generated by negative ion FAB.38 An additional fragment ion observed at miz 506 corresponds to the loss of neutral adenosine nucleoside from the ion at m/z 739. Gel slices containing 25 pg, 2.5 Kg, and 250 ng of 5’-d(AGCT)-3’were prepared and analyzed. Figure 5b shows the LD mass

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-/

z

Figure 5. (a)Standard matrix-assisted negative ion LDIFTMS spectrum of 5’d(AGCT)-3’. (b) Matrix-assisted negative ion LD/FTMS spectrum of 5’d(AGCT)-3’, obtained with the freeze-squeeze method from an agarose gel slice containing 250 ng of analyte. spectrum of 5’-d(AGCT)-3’obtained from a gelslice containing a t the maximum 250 ng of sample. The 40 pL recovered from this gel slice using the freeze-squeeze procedure was evaporated onto the probe surface. A 10-pL sample of matrix solution was then deposited and evaporated on the sample residue. Even at this low sample level, ions corresponding to 5’-d(AGCT)-3’ are easily identified in the mass spectrum.

CONCLUSIONS The results of this study indicate that the freeze-squeeze technique of recovering analyte from agarose gels, combined with matrix-assisted LD/FTMS analysis, can be a sensitive and rapid way (5-min total sample preparation time) of performing detailed characterization of selected gel bands. While direct laser desorption can also be used to obtain spectra from analyte-doped gel slices, the freeze-squeeze technique usually gave substantially better detection limits and was no more cumbersome or time-consuming than the direct desorption method. While the detection limits for this experimental approach were in the low-picomoleregion, these values are dependent on optimization of the MALDI technique for the efficient production of molecular (M - H)- ions from the oligonucleotides. Fragmentation observed in the MSiMS spectra of the analytes recovered from gel slices was sufficient to deduce the unambiguous sequence of bases in the oligonucleotides and was useful for resolving isomeric oligonucleotides. One disadvantage of the freeze-squeeze method is that it is destructive to the gel slice being examined. Because large DNA fragments (over thousands of base pairs) can be recovered from gels using this method, it appears that sample handling and the MALDIiFTMS techniques need to be optimized. For example, if all of the recovered gel fluid (41) Castoro, J. A,; Chiu, R. W.; Monnig, C. A,; Wilkins, C. L. J . A m . Chem. S o c . 1992. 114. 7571.

ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993

could be evaporated onto an area of the probe tip corresponding to the size of the laser beam (approximately 200 X 200 pm), much better signal to noise could be obtained for lower amounts of analyte. Recent developments in MALDI/ FTMS have demonstrated that this technique can be used for the off-line detection of biomolecules with molecular masses ranging from 1000 to 17000 Da which have been separated by capillary zone electrophoresis.41 The advances in extending the upper mass limit of MALDI techniques for both time-of-flight and FTMS instruments suggest that mass spectrometry may play an important role in the detection of large biomolecules separated by gel electrophoresis techniques.

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ACKNOWLEDGMENT Research sponsored jointly by the Office of Health and Environmental Research, U.S. Department of Energy under Contract DE-AC05-840R21400withMartin Marietta Energy Systems, Inc. and by the Laboratory Graduate Participation Program under Contract DE-AC05-760R00033between the US. Department of Energy and Oak Ridge Associated Universities. RECEIVED for review August 17, 1992. Accepted February 8, 1993.