Characterization of oligonucleotides and nucleic acids by mass spectrometry

Characterization of oligonucleotides and nucleic acids by mass spectrometry Patrick A Limbach, Pamela F Crain and James A McCloskey University

The

continued

refinement

ions, electrospray

of Utah, Salt Lake City, USA

of two

ionization

recent methods

and matrix-assisted

for producing

laser desorption

has resulted in new techniques

for the rapid characterization

tides

Using

by

molecular

mass

spectrometry.

mass measurements

at the 20-mer

2 Da, can now be made routinely achieved in the development

of oligonucleo-

available

level,

with

instruments,

errors

less than

in less than 15 min. Progress has also been

of mass spectrometry

oligonucleotides Current Opinion

commercially

gas-phase ionization,

for rapid sequencing

of

smaller than 25 residues.

in Biotechnology

Introduction Mass spectrometry has long played a notable role in the structural characterization of nucleic acid constituents and related compounds, although most work has been carried out at the monomer level as a result of the high polarity of nucleotides [l]. Even so, the applicability of mass spectrometry to oligonucleotides [2,3], even beyond the lOO-mer level [4,5*,6,7*,8*,9], is recent and derives largely from the discovery of electrospray ionization (ESI) [lo] and matrix-assisted laser desorption ionization (MALDI) [ 111.

Broadly, mass spectrometric measurements involve the determination of molecular mass, or of the masses of dissociation products of gas-phase ions, which can then be related to various structural properties, such as sequence. In either case, the crucial step lies in the conversion of liquid- or solid-phase solutions of the analytes into gaseous ions. ESI- and MALDI-based methods have particularly benefitted the analysis of oligonucleotides because these techniques accomplish the otherwise experimentally difficult task of producing gas-phase ions from solution species that are both very highly solvated and are highly ionic. As impressive demonstrations of these new methods continue to grow in the literature, it is not always clear whether such results “represent heroic achievements..., routine results obtainable on demand using an accessible laboratory tool, or some intermediate situation” [12*]. This situation arises because many reports from the past several years have dealt with unusually rapid technical developments as well as the demonstration of principles and measurements on

1995,

6:96-l

02

model compounds. Thus, true tests of day-to-day utility in nucleic acid chemistry, as demonstrated by published applications, have been relatively few, particularly when compared with the applications of mass spectrometry in protein chemistry [12*]. The present review represents our conclusions as to the most important developments during the past few years concerning the mass spectrometry of oligonucleotides using ES1 and MALDI. Emphasis is placed on methods that can be presently applied using conventional instruments, but coverage also includes recent reports on instrumentation and procedures that are likely to play important future roles in this rapidly developing field.

Ionization and mass analysis Electrospray ionization mass spectrometry

The electrospray process [ 10,13,14] generates multiplycharged ions from a liquid solution while it is passed through a narrow capillary into a strong electrostatic field (see Fig. 1). A quadrupole mass spectrometer is presently the mass analyzer of choice for most routine oligonucleotide analysis by ESI, and this combination has generated the vast majority of results presented to date. Currently, several variations of the electrospray ion source are available [10,15,16] that extend the solvent types and flow rates accessible to ESI, but none shows a particular advantage for oligonucleotide analysis. A relatively recent innovation has been the use of low flow-rate electrospray sources that result in reduced sample consumption (e.g. at the high femtomole to low picomole level) [17*,18*].

Abbreviations CID-collision-induced dissociation; ESl-eiectrospray ionization; FTICR-Fourier transform ion cyclotron resonance; MALDI-matrix-assisted laser desorption ionization; TEA-triethylammonium; TOF-time-of-flight.

96

0 Current Biology Ltd ISSN 0958-1669

Characterization of oligonucleotides and nucleic acids by mass spectrometry Limbach ef al.

intensity (relative units)

Mass-to-chargeratio (m/z) 0

1995 Current Opinion in Biotechnology

Matrix-assisted desorption ionization mass spectrometry

The MALDI process produces ions from an analyte cocrystallized in a matrix that absorbs at the wavelength of laser irradiation. Predominantly single-charged ions are generated following the rapid transfer of energy from the irradiated matrix to the sample (see Fig. 2), although the exact process of ionization is not well understood [19-211. Because the laser beam is pulsed, the mass analyzer of choice is a time-of-flight (TOF) design. In general, two TOF designs are used in MALDI: linear geometries and reflectron geometries. The reflectron compensates for the energy spread within the original ion beam, resulting in an improved resolution, from Intensity (relative units)

Fig. 1. ESI mass spectrum of the 76-mer tRNAval1 from Escherichia co/i acquired using a quadrupole mass analyzer [VI. A family of ions are produced, each having a different mass-to-charge ratio (m/z), which is dictated by the number of net negative charges on the ion, ranging from 20 to 29 in this case (shown in bold above peak). Each m/z value constitutes an independent measure of M,; 24 682 in this example, compared with 24 681 calculated from the known sequence.

twofold [22*] to fourfold [23**] compared with the linear analyzer. The reduced transmission effkiency of the reflectron geometry, however, results in lower sensitivity compared with the linear analyzer [22*]. Thus, trade-offs exist between these two TOF geometries.

Other instrumental designs

Both ES1 and MALDI have been used with other types of mass analyzer, including Fourier transform ion cyclotron resonance (FTICR) [24,25], sector [26], and ion-trap [27,28’] instruments. These analyzers, although commercially available in some cases, are presently used

100

WH) 75

50 (h&2H)*25

6000 0

8000

10000

12000

14000

Mass-to-chargeratio (m/z) 1995 Current Opinion in Biotechnology

Fig. 2. MALDI mass spectrum of the 40-mer d(TACACATTTTATGllTATlTATCllTGCTTTTCAAAAGGC) acquired with a linear TOF mass analyzer following ultraviolet irradiation at 337 nm. One principal ion is produced, corresponding to one net negative charge resulting from the loss of hydrogen (H) from the neutral molecule (M). The measured mass value (not reported in this case) is obtained from the peak centroid. The broad unresolved signal at the base of the peak results from MALDI-induced fragmentation, a problem which is essentially absent using the more stable RNA [7-I. (Adapted from [57*].)

97

98

Analytical biotechnology

more for research and instrumentation development, and thus the results reported should not be interpreted as routine.

Measurement of molecular mass Relative mass is an intrinsic molecular property, which when measured with high accuracy, becomes a unique and unusually effective parameter for characterization of synthetic or natural oligonucleotides. Mass spectrometry based methods can be broadly applied not only to normal (phosphodiester) nucleotides, but also to phosphorothioates [29,30’,31], methylphosphonates [32] and other derivatives [33,34].

to sample solutions can be used to suppress adventitious cations derived from solvents or matrix materials [4,8’,9,29,33,37]. In MALDI, the amount of sample used is dictated solely by how much can be easily handled because sample consumption is typically at the femtomole level or less [4] and the unused material can be recovered from the probe for further use. The choice of matrix is dictated by the laser wavelength, and although 3-hydroxypicolinic acid (for ultraviolet wavelength studies) [44] presently appears to be the most effective, the search for the optimal matrix continues.

Accuracy of mass analysis General characteristics of electrospray ionization and matrix-assisted desorption ionization mass spectra

Both DNA [2,9,13,35] and RNA [8*,36’] are readily converted to gas-phase negative ions following ESI. Although the ionization efficiency is different for DNA homopolymers (dTl@lAloaCro=dGlo) [37], both these and mixed base species are readily detectable. Suppression of ionization of a given component in a mixture as a result of the presence of other components has not been reported. Both positive [3,4,22*,23**] and negative ions [38] can be detected following MALDI. Generalizations about typical features of MALDI mass spectra of nucleic acids are not readily made, owing to the multitudes of protocols in use, such as laser wavelength (infrared versus ultraviolet, which in turn dictates matrix choice), method of sample preparation and analyzer geometry The quality of mass spectra from MALDI analyses is profoundly influenced by base composition. Thus, polythymidylic acids are readily analyzed [4,39-421, but polydeoxyguanylic acids are a distinct challenge and mixed base oligomers represent an intermediate situation. These findings are attributed to the fragmentation of guanosine residues in DNA [4,6,40,42], a process which has been shown to occur in the gas phase [5*] and not on the target before desorption. As a consequence of the increased stability of the glycosidic bond in RNA, it is more readily analyzed than DNA [5’]. I n mixtures of mixed-base deoxyoligomers (e.g. the products of Sanger sequencing reactions), smaller oligomers can suppress ionization of larger ones when all are present in equimolar amounts [431*

Sample preparation

The crucial consideration for nucleic analysis by either MALDI or ES1 is that the sample be as fi-ee as possible of involatile cations (e.g. Na+ and Mg2+), which severely limit both the accuracy of molecular mass determination and ion yields for larger oligonucleotides. Ammonium (or tetraethylammonium) are preferred salt forms for both ES1 [35] and MALDI [4] analyses. Analogously, ammonium and tetraethylammonium additives

Accuracy of molecular mass measurement is a function of both the ionization method and mass analyzer. In general, ES1 results have been more routinely accurate than those obtained using MALDI, primarily for two reasons. First, multiple charging produces a family of ions, each of which provides an independent measure of molecular mass, the average of which improves the overall precision and accuracy [45]. Also important is the inherent improvement in resolution attainable using the quadrupole mass spectrometer compared with the combined technique of MALDI-TOE Mass measurement errors of 0.03% and lower are typical [35,46**], and with appropriate sample clean-up, values lower than 0.01% can be obtained routinely [8*,9]. In contrast, for MALDI, typical measurement errors are between 0.03% and 0.05% [4,22*], and with use of internal standards, mass accuracies of the order of 0.01% can be obtained for small oligonucleotides [5*,30*]. Unfortunately, mass errors for larger oligonucleotides are higher (e.g. 0.3% for tRNA [4]) as a consequence of the lower inherent resolution of the TOF mass analyzer. Ions horn adducts and base losses cannot be resolved from the molecular ion and will influence peak centroid measurement.

Molecular size limits

ES1 has been employed to measure the mass of oligonucleotides as large as 76-87-mers (including intact tRNAs) [8*,9,35], and some papers even report the analysis of 120-130-mers [8’,9]. The size limit for quadrupole analyzers, using ESI, is around a 300-mer because of peak overlap between ions differing by one charge. With the TOF analyzer, no theoretical upper mass limit exists, thus, practical aspects of producing and detecting large ions are the factors that dictate the ultimate limit. It appears that for oligodeoxynucleotides, resolution and mass accuracy are severely restricted above the 30-mer level as a result of dissociation reactions [5*,22*]. In contrast, oligoribonucleotides f?om 76-120mers (including intact tRNAs and 5s rRNA) have been mass-analyzed [4,5*,22*,44]. Recently, a synthetic 461nucleotide RNA was successfully vaporized and detected using MALDI [7*].

Characterization of oligonucleotides and nucleic acids by massspectrometry Limbach et al. Determination modification

of nucleotide

sequence and

sites

The utility and limitations of accurate mass measurement for direct determination of the base composition of oligonucleotides, including modifications, have recently been discussed [8*,47*]. For larger oligonucleotides, accurate mass measurement can be used in combination with limited sequence data to constrain the number of structural candidates [8*,25].

Mapping of modification sites in nucleic acids

A method has been developed for the characterization of modifications in RNA [36*], and this has been applied to both tRNA [48] and 5s rRNA [49]. The strategy capitalizes on the high accuracy with which oligonucleotide mass can be measured using ESI, such that base composition can be determined [47*]. When this approach is used in conjunction with liquid chromatography mass spectrometry data [50], modifications can be completely characterized at the nucleoside level and assigned to oligonucleotides defined by selective RNase cleavage.

Enzyme-based sequencing methods

Mass spectrometry can be used to measure the molecular weights of oligonucleotides in mixtures generated by sequential removal of 3’ or 5’ terminal nucleotides by phosphodiesterase II or I, respectively. The resulting ‘mass ladder’ can then be used to reconstruct the sequence, using either MALDI [7*,33] or ES1 [51]. The impetus for many studies of MALDI analysis of oligonucleotides has been its potential use in largescale gene sequencing efforts. Determination of molecular weights of chain-termination products in the sequencing reaction mixture obviates the requirement for electrophoretic separation. Many MALDI studies have been carried out on synthetic oligonucleotide mixtures (e.g. [22*,29,30’,43,44,52]), but practical application to ‘real’ samples (e.g. those containing template, primer, proteins, salts and dNTPs) has not been reported as yet. A number of obstacles presently render MALDI noncompetitive with various existing techniques in routine use [53], including time-consuming sample clean-up, lack of sensitivity [6,29,43], inability to detect ‘large’ fragments in the presence of small ones [43] and general instability of DNA under MALDI conditions [5*]. Copying of the DNA to be sequenced into RNA for MALDI analysis may obviate the problem of detection of long DNA strands, but this proposal [7*] has not yet been developed.

Sequencing by gas-phase dissociation

One of the most promising methods for sequencing oligonucleotides below the -25-mer level involves the mass analysis of hagment ions resulting Tom backbone cleavage [l], which are then used to construct a ‘mass ladder’. Data acquisition is rapidusually a matter of

seconds and thus, much shorter than sample preparation time -and can be used both for simultaneous bi-directional sequencing and for recognition of base, sugar [54] or phosphate [55] modification. In the case of phosphodiester modification in which no net negative charge is involved, the molecule can be analyzed in the positive ion mode, as clearly demonstrated for methylphosphonates [46.*]. In general, the method by which fragment ions are generated for this purpose is tied to the type of instrument used and is accomplished either by collision-induced dissociation (CID) of ESIgenerated ions (e.g. quadrupole [46**], ion-trap [27,28*], or FTICR instruments [25]) or by spontaneous dissociation, following MALDI with both FTICR [56] and TOF [57*] instruments. The problem of correct peak assignment (see e.g. [28*,58]) is &r more acute in the case of ES1 spectra because of the multiplicity of charge states observed for any given ion. Mechanisms of dissociation have been considered in sufficient detail that sequenceion assignments can be reliably made for the purpose of sequence verification. Although optimism is warranted for the achievement of direct sequencing of oligonucleotides by mass spectrometry within the next year or so, it has not yet been demonstrated that complete sequences of structurally unknown oligonucleotides can be routinely determined. Detection

of non-covalent

interactions

The electrospray process is sufficiently gentle that, under some experimental conditions, non-covalently bound species present in solution can be transferred to the gas-phase and observed as stoichiometrically defined ion complexes [59**]. Important examples include gas-phase duplexes of complementary 8-mers [60], 20-mers [61] and the non-covalent tetraplex d(CGCG4GCG)J, which was observed as a gas-phase ion in the presence, but not absence, of Na+ [62], in concert with previously established solution-phase properties. In addition, a recent report has studied the interaction of actinomycin D with single-stranded oligodeoxynucleotides [63*]. The above studies generally mirror a larger body of analogous work on proteins [12*]. One goal of this promising work is to develop the ability to use gas-phase data (determined by mass spectrometry) to study solution-phase interactions. Even so, care must be taken in the interpretation of such data at this early stage of development. Conclusions The ionization techniques of ES1 and MALDI have opened new doors for the analysis of oligonucleotides and their analogs by mass spectrometry. At this intermediate stage of rapid development of instrumentation and methodology, neither technique is clearly preferable for general use. Each technique exhibits its own characteristics and limitations, resulting in part from the mass analyzers commonly coupled with each ionization method. Direct sequencing by mass spectrometry is useful for verification of sequence, but some cau-

99

100

Analytical biotechnology

tion should be exercised over the endorsement of mass spectrometry as a potential method for large-scale genomic sequencing [40,52,64]. Although applications to more modest levels of DNA sequencing are possible in the near future, no present evidence suggests that MSbased techniques will become competitive with existing techniques in routine use [53]. Prospects are, however, bright for continued progress in several rapidly evolving areas. Notable among these are the following: the development of mass analyzers capable of highly accurate mass measurement [24], especially over the lOO-mer level; the direct analysis of mixtures of oligonucleotides by direct chromatography/electrophoresis mass spectroscopy interfacing [65]; further exploratory studies of non-covalent associations in the gas phase [66]; and applications to nucleic acid and protein-nucleic acid [67*] crosslinking studies. Acknowledgements The authors are pleased to acknowledge the contributions of various colleagues who provided copies of manuscripts before publication, and for the support of the National Institute of General Medical Sciences (GM21 584).

References and recommended Papers of particular interest, published review, have been highlighted as: . of special interest .. of outstanding interest

8. .

Limbach PA, Crain PF, McCloskey IA: Molecular mass meac urement of intact ribonucleic acids via electrospray ionization quadrupole mass spectrometry. J Am Sot Mass Spectrom 1995, 6:27-29. An investigation of sample clean-up procedures required to reduce the level of cation adducts (e.g. Na+ and MgZ+J for the accurate mass analysis of large oligonucleotides. This paper also discusses the potential utility of mass analysis of small nucleic acids, in particular tRNA. 9.

Potier N, Van Dorsselaer A, Cordier Y, Roth 0, Bischoff R: Nezative electrospray ionization mass spectrometry of synthe& and chemically modified oliRonucleotides. Nucleic Acids Res 1994, 2233895-3903. -

10.

Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse Electrospray ionization for mass spectrometry of biomolecules. Science 1989, 246:64-71.

11.

Karas M, Hillenkamp F: laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem 1988, 60:2299-2301.

12. .

Burlingame AL, Boyd RK, Caskell SC: Mass spectrometry. Anal Chem 1994, 66:634R-683R. Although this biennial review on mass spectrometry excludes nucleic acids this time round, it does provide excellent coverage of ESI, MALDI and the associated mass analyzers, all of which have undergone rapid development over the past several years. 13.

Smith RD, Loo ]A, Edmonds CC, Barinaga CJ, Udseth HR: New developments in biochemical mass spectrometry: electrospray ionization. Anal Chem 1990, 62:882-899.

14.

Kebarle P, Tang L: From ions in solution to ions in the gas phase - the mechanisms of electrospray mass spectrometry. Anal Chem 1993, 65:972A-986A.

15.

Bruins AP, Covey TR, Henion ID: Ion spray interface for combined liquid chromatography/atmospheric pressure ionization mass spectrometry. Anal Chem 1987, 59~2642-2646.

16.

Banks JF Jr, Shen 5, Whitehouse CM, Fenn JB: Ultrasonically assisted electrospray ionization for K/MS determination of nucleosides from a transfer RNA digest. Anal Chem 1994, 66:406-414.

reading within

CM: large

the annual period of

1.

Crain PF: Mass spectrometric techniques in nucleic acid research. Mass Spectrom Rev 1990, 9:505-554.

2.

Covey TR, Bonner RF, Shushan BI, Henion J: The determination of protein, oligonucleotide and peptide molecular weights by ion-spray mass spectrometry. Rapid Commun Mass Spectrom 1988, 2:249-256.

17. .

3.

Spengler B, Pan Y, Cotter R: Molecular weight determination of underivatized oligodeoxyribonucleotides by positive-ion matrix-assisted ultraviolet laserdesorption mass spectrometry. Rapid Commun Mass Spectrom 1990, 4:99-l 02.

Emmett MR, Caprioli RM: Micro-eledrosnrav mass swctrometry: ultra-high-sensitivity analysis of pe&l& and pioteins. / Am Sot Mass Spectrom 1994, 5:604-613. This paper and [17*] describe a new instrumental source design that both dramatically reduces sample consumption rate in electrospray and pro vides sensitivities comparable to MALDI. This source is not yet available commercially, although the modifications required for its construction are relatively minor.

4.

Nordhoff E, lngendoh A, Cramer R, Dverberg A, Stahl Et, Karas M, Hillenkamp F, Crain P: Matrix-assisted laser desorption/ionization mass spectrometry of nucleic acids with wavelengths in the ultraviolet and infrared. Rapid Commun Mass Spectrom 1992, 6:771-776.

Gale DC, Smith RD: Small volume and low flow-rate electrospray ionization mass spectrometry of aqueous samples. Rapid Commun Mass Spectrom 1993, 7:1017-l 021. See [18*1 annotation. 18.

.

19.

Hillenkamp F, Karas M: Mass spectrometry of peptides and proteins by matrix-assisted ultraviolet laser desorption/iinization. In Methods Enzymol, vol 193. Edited by McCloskey ]A. San Diego: Academic Press; 1990:281X295.

20.

Hillenkamp F, Karas M, Beavis RC, Chait BT: Matrix-assisted laser desorption/ionization mass spectrometry of biopolymen. Anal Chem 1991, 63:1193A-1203A.

21.

Ehring H, Karas M, photochemistry in and relevance for mass spectrometry.

5. .

Nordhoff E, Cramer R, Karas M, Hillenkamp F, Kirpekar F, Kristiansen K, Roepstorff P: Ion stability of nucleic acids in infrared matrix-assisted laser desorption/i&ation mass spectrometry. Nucleic Acids Res 1993, 21:3347-3357. A detailed description of the use of infrared lasers for MALDI analysis of oligonucleotides. Most importantly, this paper contains a good discussion, with examples, of the greater gas-phase stability of ribonucleotides compared with deoxyribonucleotides. 6.

7. .

Tang K, Allman SL, Chen CH: Matrix-assisted laser desorption ionization of oligonucleotides with various matrices. Rapid Commun Mass Spectrom 1993, 7:943-948.

Kirpekar F, Kristiansen K, Roepstorff P, Nordhoff E, Lezius A, Hahner 5, Karas M, Hillenkamp F: Matrix assisted laser desorp tion/ionization mass spectrometry of enzymatically synthesized RNA up to 150 kDa. Nucleic Acids Res 1994, 22:3866-3870. Mass spectra of RNA up to the 461-mer level are successfully obtained, clearly verifying earlier conclusions that larger ribonucleotides are not subject to the same factors that cause degradation of deoxyribonucleotides following laser desorption. This paper also provides striking evidence that the size limit for production and detection of gas-phase RNA ions has not yet been reached.

Hillenkamp F: Role of photoionization and ionization processes of organic molecules matrix-assisted laser desorption ionization 0% Mass Spectrom 1992, 27:472-480.

22. .

Wu KJ, Shaler TA, Becker CH: Time-of-flight mass spectrometry of underivatized single-stranded DNA oligomers by matrixassisted bser desorption. Anal Chem 1994, 66:1637-l 645. Provides a comparison of ion polarity (positive versus negative ion detection) for MALDI analysis of oligonucleotides. Also contains a comparison of linear and reflectron TOF geometries, with some discussion of the relative merits of the linear geometry. 23. ..

Nordhoff E, Kirpekar F, Karas M, Cramer R, Hahner 5, Hillenkamp F, Kristiansen K, Roepstorff P, Lezius A: Comparison of IR- and UV-matrix-assisted laser desorption/ionization

Characterization of oligonucleotides and nucleic acids by mass spectrometry mass spectrometry of oligodeoxynucleotides. Nucleic Acids Res 1994, 22:2460-2465. A more detailed study of the mass spectrometry of oligonucleotides than 141, with investigations into ion polarity (positive versus negative ion detection) and linear versus reflectron TOF geometry. Data presented are some of the best oligonucleotide results to date using MALDI-TOF. 24.

Buchanan MV, Hettich tromctry of high-mass 65:245A-259A.

25.

Little DP, Chorush RA, Speir JP, Senko MW, Kelleher NL, McLafferty Fw: Rapid sequencing of oligonucleotides by high-resolution mass spectrometry. 1 Am Chem Sot 1994,116:4893-4897.

26.

27.

RL: Fourier-transform biomolecules.Anal

mass specChem 1993,

McEwen CN, Larsen 6s: Accurate mass measurement of proteim using electrospray ionization on a magnetic sector instrument. Rapid Commun Mass Spectrom 1992, 6:173-l 78. McLuckey SA, Van Berkel CJ, Glish CL: Tandem mass spectrometry of small, multiply charged oligonucleotides. 1 Am Sot Mass Spectrom 1992, 3:61%70.

McLuckey SA, Habibi-Coudarzi S: Decompositions of multiply charged oligonucleotide anions. 1 Am Chem Sot 1993, 115:12085-12095. This report represents the most complete study, thus far, of the pathways and mechanisms of oligonucleotide dissociation, providing and extending the groundwork for the determination of sequence from mass spectra. These studies were carried out using ESI with an ion trap mass analyzer. The results should be largely applicable to other mass analyzers, although the extent to which the mechanisms of dissociation reactions may differ as a result of different experimental configurations is not yet known. Currie CJ, Yates JR Ill: Analysis of oligodeoxynucleotides by negative-ion matrix-assisted laser desorption mass spectrometry. / Am Sot Mass Spectrom 1993, 4:955-963.

37.

Bleicher K, Bayer E: Various factors influencing the signal intensity of oligonucleotides in elect-ray mass spectrometry. Biol Mass Spectrom 1994, 23:320-322.

38.

Hillenkamp F, Karas M, lngendoh A, Stahl B: Matrix-assisted UV laser desorption/ionizatio: a new approach to mass spectrometry of large biomolecules. In Biological mass spectromerry. Edited by Burlingame AL, McCloskey ]A. Amsterdam: Elsevier Science Publishers; 1990:49-60.

39.

Huth-Fehre T, Cosine JN, Wu KJ, Becker CH: Matrix-assisted laser desorption mass spectrometry of oligodeoxythymidylic acids. Rapid Commun Mass Specrrom 1992, 6:209-213.

40.

Parr CR, Fitzgerald MC, Smith LM: Matrix-assisted laser desotption/ionization mass spectrometry of synthetic oligodeoxyribonucleotides. Rapid Commun Mass Spectrom 1992, 6:369372.

41.

Tang K, Allman SL, Jones RB, Chen CH, Araghi S: laser mass spedrometry of polydeoxyribothymidylic acid mixtures. Rapid Commun Mass Spectrom 1993, 7:63-66.

42.

Schneider K, Chait BT: Matrix-assisted laser desorption mass spectrometry of homopolymer oligodeoxyribonucleotides. Influence of base composition on the mass spectrometric response. Org Mass Spectrom 1993, 28:1353-1361.

43.

Fitzgerald MC, Zhu L, Smith LM: The analysis of mock DNA sequencing reactions using matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 1993, 71895-897.

44.

Wu KJ, Steding A, Becker CH: Matrix-assisted laser desorption time-of-flight mass spectrometry of oliganucleotides using 3. hydroxypicolinic acid as an ultraviolet-sensitive matrix. Rapid Common Mass Spectrom 1993, 7:142-146.

45.

Mann M, Meng CK, Fenn JB: Interpreting mass spectra of multiply charged ions. Anal Chem 1989, 61:1702-l 708.

30. .

Wang BH, Biemann K: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of chemically modified oligonucleotides. Anal Chem 1994, 66:1918-l 924. An investigation of the use of MALDI for accurate mass analysis of small oligonucleotides, with an emphasis on determining modification mass and distinguishing between uridine- and @dine-containing oligonucleotides. 31.

Hathaway GM: Characterization of modified oxyoligonucleotides by MALDI, time-of-flight etry. Biotechniques 1994, 17:150-l 55.

and normal demass spectrom-

32.

Keough T, Baker TR, Dobson RLM, Lacey MP, Riley TA, Hasselfield JA, He&berth PE: Antisense DNA oligonucleotides II: the use of matrix-assisted laser desorption/ionization mass spectrometry for the sequence verifica&n of methylphosphonate oligodeoxyribonucleoGdes. Rapid Commun Mass Spectram 1993, 7:195-200.

33.

Pieles U, Ziircher W, Sch;ir M, Moser HE: Matrix-assisted laser desorotion ionization time-of-fliaht mass swctrometrv: a powetful’tool for the mass and se&ence anilysis of naiUral and modified oliRonucleotides. Nucleic Acids Res 1993, 21:3191-3196. -

34.

Fitzgerald MC, Parr CR, Smith LM: Basic matrices for the matrix-assisted laser desorption/ionization mass spectrometry of proteins and oligonucleotides. Anal Chem 1993, 65:3204-3211.

35.

Stults JT, Marsters JC: Improved electrospray ionization of synthetic oligodeoxynucleotides. Rapid Commun Mass Specrrom 1991, 5:359-363.

Kowalak JA, Pomerantz SC, Crain PF, McCloskey JA: A novel method for the determination of posttranscriptional modiication in RNA by mass spectrometry. Nucleic Acids Res 1993, 21~4577-4585. This paper describes a novel method for measuring molecular masses of oligonucleotides produced by RNase Tl hydrolysis of RNA, with errors of less than 1 Da. The technique directly provides base composition and recognition of modification from incremental increases in mass (e.g. 14Da for one methyl group). Taking advantage of the extensive availability of rRNA gene sequences, specific sequence sites or regions in large RNAs (e.g. 165 and 23s rRNA) can be targeted for cleavage and mass analysis. The principles described are also applicable to other co-

et al.

valent modifications that are expressed as changes in mass, such as those resulting from photochemical cross-linking or xenobiotic damage.

28. .

29.

Limbach

46. ..

Baker TR, Keough T, Dobson RLM, Riley TA, Hasselfield JA, Hesselberth PE: Antisense DNA oliRonucleotides I: the use of ionspray tandem mass spedrometh for the sequence verification of methylphosphonate oligodeoxyribonucleotides. Rapid Commun Mass Spectrom 1993, 7:190-194. In this paper, the sequence of an 18-mer is verified by product ion scans of multiply charged parents. Although demonstrated on methylphosphonates in positive-ion mode, the experimental procedures are all relevant to negative-ion mode ESI of oligonucleotides. This report was one of the earliest to demonstrate the power of CID to verify sequence information and is one of the few that has analyzed ‘real world’ samples. 47. .

Pomerantz SC, Kowalak )A, McCloskey JA: Determination of oligonucleotide composition from mass spectrometrically measured molecular weight. j Am Sot Mass Spectrom 1993, 4:204-209. Describes a mass spectrometric technique for determining the base composition of an oligonucleotide. If the compositional value of any one nucleotide is known (e.g. from selective chemical modification or nuclease cleavage) oligonucleotide composition can be determined solely from mass spectometrically measured molecular mass. 48.

Kowalak JA, Dalluge JJ, McCloskey IA, Stetter KO: Role of posttranscriptional modification in stabilization of transfer RNA from hyperthermophiles. Biochemistry 1994, 33:7869-7876.

49.

Bruenger E, Kowalak JA, Kuchino Y, McCloskey )A, Mizushima H, Stetter KO, Crain PF: 5S rRNA modification in the hyperthermophilic archaea Su/folobus solfataricus and Pyrodictium occulturn. FAX6 / 1993, 7:196-200.

50.

Pomerantz SC, McCloskey JA: Analysis of RNA hydrolyzates by K/MS. In Methods enzymology, vol 193. Edited by McCloskey )A. San Diego: Academic Press; 1990:796-824.

51.

Limbach PA, McCloskey JA, Crain PF: Enzymatic sequencing of oligonucleotides with electrospray mass spectrometry. Nucleic Acids Res Symp Ser 1994, 31:127-128.

52.

Tang K, Allman SL, Chen CH: Mass spectrometry of laser-desorbed oligonucleotides. Rapid Commun Mass Spectrom 1992, 6~365-368.

36. .

101

102

Analytical biotechnology 53.

Automa& DNA sequencing and analysis. Edited by Adams M, Fields C, Winter JC. San Diego: Academic Press; 1994.

54.

Phillips DR, McCloskey JA: A comprehensive study of the low energy collision-induced dissociation of dinucleoside monophosphates. Int J Mass Specwom /on Processes 1993, 128:61-82.

55.

56.

Hettich R, Buchanan MV: Structural characterization of normal and modified oligonucleotides by matrix-assisted laser desorption Fourier transform mass spectrometry. / Am Sot Mass Specrrom 1991, 2:402-412. Stemmler EA, Hettich RL, Hurst CB, Buchanan MV: Matrix-assisted laser desorption/ionization Fourier-transform mass spectrometry of oligodeoxyribonucleotides. Rapid Commun Mass Spectrom 1993, 7:828-836.

Nordhoff E, Karas M, Cramer R, Hahner S, Hillenkamp F, Lezius A, Kirpekar F, Kristiansen K, Muth J, Meier C, Engels JW: Direct mass spectrometric sequencing of low pmol amounts of oligodeoxynucleotides with up to 21 bases by matrix-assisted laser desorption/ionization mass spectrometry. Eur / Biochem 1995, in press. Ultraviolet and infrared MALDI have demonstrated their potential for accurate and sensitive mass determination of oligonucleotides. For the analysis of larger nucleic acid sequences, metastable molecule-ion fragmentation is the main limitation in both desorption schemes. Fragment ions from additional ‘prompt’ decays, observed only in infrared-MALDI, offer structural data, which allow the derivation of sequence information from low picomole amounts of oligodeoxynucleotides, with up to 21 residues from a single mass spectrum. This paper provides two examples demonstrating the feasibility of this new sequencing technique. A model is introduced and discussed, which proposes a reaction scheme for the observed fragment-ion patterns of nucleic acids and differentiates ‘prompt’ and metastable fragmentation mechanisms. This paper also considers the role of fragmentation both in direct mass spectrometric sequencing schemes and as a limitation for the accessible mass range in nucleic acid MALDI mass spectroscopy. 57. .

58.

Crain PF, Cregson JM, McCloskey JA, Nelson CC, Peltier JM, Phillips DR, Pomerantz SC, Reddy DM: Characterization of post-transcriptional modification in nucleic acids by tandem mass spectrometry. In Mass spectrometry in the biological sciences. Edited by Eurlingame AL, Carr S. New York: Humana Press; 1995:in press.

Smith RD, Light-Wahl KJ: The observation of non-covalent in. teractions in solution by electrospray ionization mass spectrometry: promise, pitfalls and prognosis. Biol Mass Spec~rom 1993, 22:493-501. This is an excellent and perceptive review on ESI mass spectrometry. A discussion of a number of topics of importance is provided, including models of electrospray ion formation under current consideration, interface conditions that influence formation of non-covalent associations, and experimental factors used to distinguish specific from non-specific associations. 59. ..

60.

Ganem B, Li Y-T, Henion JD: Detection of oligonucleotide duplex forms by ion-spray mass spectrometry. Tetrahedron Len 1993, 34: 1445-l 448.

61.

Light-Wahl KJ, Springer DL, Winger BE, Edmonds CC, Camp DC II, Thrall BD, Smith RD: Observation of a small oligonucleotide duplex by electrospray ionization mass spectrometry. / Am Chem Sot 1993, 115:803-804.

62.

Gocdlett DR, Camp II DC, Hardin CC, Corregan M, Smith RD: Direct observation of a DNA quadruplex by electrospray ionization mass spectrometry. Biol Mass Speckom 1993, 22:181-183.

63. .

Hsieh YL, Li Y-T, Henion JO, Ganem B: Studies of non-covalent interactions of actinomycin D with singlestranded oligodeoxynucleotide by ion spray mass spectrometry and tandem mass spectrometry. Biol Mass Speclrom 1994, 231272-276. The selectivity of interaction between actinomycin D and single-stranded oligodeoxynucleotides is studied as a function of drug structure, oligonucleotide sequence and pH. This paper is a good example both of exploratory work in this area and of how experiments involving small molecule-DNA interactions might be carried out using mass spectrome-

try. 64.

Williams P: Time of flight mass spectrometry of DNA laser-ah lated from frozen aqueous solutions: applications to the Human Cenome Project. Int J Mass Spec&om /on Processes 1994, 131:335-344.

65.

Hofstadler SA, Wahl JH, Bruce JE, Smith RD: On-line capillary electrophoresis with Fourier transform ion cyclotron resonance mass spectrometry. / Am Chem Sot 1993, 115:69834984.

66.

Juhasz P, Biemann K: Mass spectrometric molecular weight determination of highly acidic compounds of biological significance via their complexes with basic polypeptides. Proc Nat/ Acad Sci USA 1994, 91:4333-4337.

67. .

Jensen ON, Barfosky DF, Young MC, von Hippel PH, Swenson S, Seifried SE: Direct observation of UV-crosslinked proteiHucleic acid complexes by matrix-assisted laser desorption ionization mass spectrometry. Rapid Commun Mass Specrrom 1993, 7:496-501. MALDI-TOF mass spectrometry is used for characterization of UVinduced protein-nucleoside crosslinks between phage T4 gene 32 protein and @T)zo, and between E. co/i transcription termination factor rho and 4.thiouridine diphosphate. This excellent paper clearly demonstrates the potential of mass spectrometry in protein-nucleic acid crosslinking studies.

PA Limbach, PF Gain and JA McCloskey, Department of Medicinal Chemistry, 3llA Skaggs Hall, University of Utah, Salt Lake City, Utah 84112, USA.