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The essence of DNA sample preparation for MALDI mass spectrometry
J. Biochem. Biophys. Methods 70 (2007) 311 – 318 www.elsevier.com/locate/jbbm
Review
The essence of DNA sample preparation for MALDI mass spectrometry Sascha Sauer ⁎ Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics (Prof. H. Lehrach), Ihnestrasse 73, D-14195 Berlin, Germany Received 26 May 2006; accepted 16 October 2006
Abstract Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important analytical technique in nucleic acid research. MALDI is used for quality control of oligonucleotides as well as for analyzing DNA markers. Sample preparation of nucleic acids is crucial for obtaining high-quality mass spectra. Sample purity, solvent content, suitable matrices, and substrate surfaces, as well as laboratory conditions affect spectra quality. This review presents essential information with regard to sample preparation, DNA modification chemistry, and DNA purification, along with a discussion of instrumental advances, which facilitate and extend the applicability of MALDI in genomics. © 2006 Elsevier B.V. All rights reserved. Keywords: MALDI; DNA; Oligonucleotide; Matrix; Purification; SNP
Contents 1.
Introduction . . . . . . . . . . . 1.1. Instrumental aspects . . . 1.2. Matrices . . . . . . . . . 1.3. Chemical modification of 1.4. Preparation methods . . . 1.5. Purification procedures . 1.6. Outlook . . . . . . . . . 2. Note added in proof . . . . . . Acknowledgements . . . . . . . . . References . . . . . . . . . . . . . .
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1. Introduction Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has become a powerful tool in life science (Fig. 1) [1]. MS detects the mass-to-charge ratio of analyte ions either in positive- or in negative-ion mode thus, no labelling and signal clustering, as necessary for fluorescence detection, are required [2]. In MALDI, co-crystals formed by small and acidic organic molecules, termed the “matrix”, and biological com⁎ Tel.: +49 30 84131565; fax: +49 30 84131365. E-mail address: [email protected]. 0165-022X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2006.10.007
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pounds, such as peptides or nucleic acids, are irradiated with a pulsed laser at a wavelength close to a resonant absorption band of the matrix molecules. This induces an energy transfer and desorption process, evaporating analyte and matrix molecules into the gas phase, which is a high vacuum. During MALDI a proton-transfer reaction of matrix and analyte molecules in the gas phase is induced, predominantly leading to singly charged ions [3]. The ions are accelerated by an electric field and guided by ion optics into a mass analyzer. MALDI-MS can be efficiently applied in quality control of oligonucleotides, analyzing markers such as single nucleotide polymorphisms (SNPs) and cytosine methylation, as well as for
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Fig. 1. The principle of MALDI-MS is illustrated. Irradiation of co-crystals formed by organic molecules, called the “matrix”, and oligonucleotides with a pulsed ultraviolet (UV-) laser at a wavelength close to a resonant absorption band of the matrix induces an energy transfer and desorption process, evaporating analyte and matrix molecules into the gas phase (vacuum: ca. 10− 7 mbar). Common UV-lasers are either nitrogen lasers that emit at 337 nm or frequency-tripled Nd:YAG lasers emitting at 355 nm. The photon energy of these UV lasers is in the range of about 3.49–3.68 eV, and the photon width is about 1–5 ns. During the desorption process and/or in the gas phase, ionization of matrix and analytes takes place by proton-transfer reactions. Product ions are accelerated by an electric field. In general, MALDIMS is executed with time-of-flight (TOF) separation: ions are guided by ion optics into a field-free flight-tube (vacuum: ca. 10− 9 mbar) and separated according to their mass-to-charge ratios before they impinge on a micro-channel plate detector (MCP). The scale of this figure is not proportional to the instrumental set-up of common MALDI mass spectrometers.
analysis of allele-specific expression and detection of alternative splicing [4–6]. Most of these approaches involve buffered solvents and a certain amount of salts in reaction solutions. Sample preparation of DNA is important for obtaining highquality MALDI mass spectra [7]. Sample purity, solvent content, substrate surface and laboratory conditions, such as humidity and temperature, affect matrix/analyte co-crystallization. Nucleic acids are harder to detect by MALDI than peptides. Size-dependent fragmentation of DNA during MALDI is a common phenomenon caused by excess energy during laser desorption, which results in a loss of signal intensity for intact DNA. Fragmentation can be induced by protonation of DNA bases, leading to base loss (mainly depurination) [8]. Interestingly, RNAs are more stable in MALDI due to the additional 2′hydroxyl group, which stabilizes the glycosidic bond, leading to significantly reduced depurination and fragmentation of the whole oligomer [9]. An additional limiting factor may be a general bias of MALDI towards smaller DNAs [10]. The sensitivity of commercially available MALDI mass spectrometers is presently in the range of ca. 1 fmol–100 amol per analyte. Modern MALDI mass spectrometers generally use delayed extraction [11], which enables high resolution of mass signals, allowing the distinction of oligonucleotides in the mass range of 1000–10,000 Mr. These instruments are capable of recording at least one spectrum per second, and multiple nucleic acids can be detected in a single measurement (multiplex analysis). Additionally, relative quantification of different nucleic acids is feasible [12]. This can be attributed to the chemical nature of nucleic acids, which, in contrast to peptides, are rather monotonous polymers, practically displaying no differences during laser desorption. The biggest problem in the detection of nucleic acids by MALDI consists in their negatively charged sugar-phosphate backbones [7]. Phosphate residues provide a site of negative charge in solution. Each DNA oligomer carries as many negative charges as phosphate linkages. The affinity of the phosphates for
alkali counterions, such as sodium and potassium, is high, but does not result in complete saturation. Thus these ions interfere with the ionization process, by inducing adducts, and this severely limits signal intensity and quality. The use of ammonium salts in MALDI is a well-known approach to counteract ion affinities [13]. In solution, ammonium exits as an ammonium ion, whereas in the gas phase ammonium is readily lost, resulting in a reduced adduct structure of nucleic acids. However, ammonium ions introduce a degree of suppression to the desorption process, which decreases analytical resolution and sensitivity. Furthermore, residual amounts of denaturants, such as urea or guanidineHCl and detergents, such as Tween-20 or Triton X, interfere with matrix crystallization and decrease or even eliminate signals in MALDI-MS. Purification methods must be applied prior to MALDI to solve the above-mentioned problems with impurities, which are often associated with biological assays. 1.1. Instrumental aspects UV-laser MALDI-MS (Fig. 1) has not only been used for the analysis of oligonucleotides or primer extension products of SNPs with a size of about 5000–8000 Mr, but also for complex mixtures containing large DNA fragments [7] such as DNA sequencing ladders. In general, these products are routinely detected in gene laboratories by capillary electrophoresis [14]. The difficulty of measuring many DNA fragments consists in the amount of ion current available to simultaneously detect different oligonucleotides and the effects this implies on the detection sensitivity and resolution of these analytes. Moreover, due to the abovementioned loss of signal intensity and mass resolution with increasing size of nucleic acids this mass spectrometric technology is limited to nucleic acids smaller than ca. 100 nucleotides. Masses as small as 9 Mr — which is the mass difference between thymine and adenine — are virtually impossible to resolve at ca. 30,000 Mr by conventional UV-MALDI. Hillenkamp and coworkers [15] have introduced infrared lasers, such as ER:YAG
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lasers, emitting at 2.94 μm with a pulse width at 85 ns, for detection of large nucleic acids, using glycerol as matrix. One problem in the MALDI analysis of large biological molecules is the sensitivity of the micro-channel plate detectors commonly used in most MALDI mass spectrometers, which drops down with increasing particle mass. The peak-width of large DNA fragments of about several hundred bases, such as PCR products, can range over several 100 Mr, and the mass accuracy is quite poor. Cryogenic detectors might be a solution to overcome this problem, but these instruments are presently far from mature enough for routine application [15]. Moreover, inherently complex signals of macromolecules result from atomic isotopes; in the case of DNA this problem could be counteracted by using expensive isotope-free nucleotides. 1.2. Matrices Matrices for MALDI are applied on evenly flat conductive plates that are introduced in the ionization chamber of the mass spectrometer. Stainless steel, aluminum/nickel alloys, and gold, or semi-conductors, such as silicon, are most commonly used plate materials. In general, the optimization of MALDI depends on identifying the best matrix and preparation method for a particular analyte. The mechanism of MALDI is not well understood and the interaction of DNA with a matrix during the desorption/ionization process eludes further investigation. However, empirical findings have aided the method. MALDI sample preparation is generally performed with acidic matrices, and acidic conditions are encountered during desorption and ionization. MALDI matrices must meet a number of requirements: (1) they must be able to embed analyte molecules, (2) they must be soluble in the same solvents as the analytes, (3) they have to be stable in vacuum, (4) they should induce co-desorption of analytes, and (5) they should promote ionization of analytes. In general, the matrix-to-analyte mixtures are in the range from 1000:1 to 10,000:1, but this can vary, depending on the preparation. Common matrices for the detection of nucleic acids are listed in Table 1.
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As mentioned above, fragmentation of DNA, frequently observed in MALDI, results from excess energy, deposited during laser irradiation. A detectable degree of depurination has been observed for large DNAs. Fragmentation mainly depends on the nature of the matrix used. A remarkable dependence of the matrix-to-DNA ratio on fragmentation was observed [8]. MALDI matrices, such as 2,3,4- or 2,4,6-trihydroxyacetophenone [16] are termed ‘hot’ if they result in excessive fragmentation, and therefore they are suitable only for small oligonucleotides. Comparatively ‘cool’ matrices, such as 3hydroxypicolinic acid [17] and picolinic acid, as well as 6-aza-2thiothymine [18], result in very little fragmentation and are also suitable for larger nucleic acids. Addition of a sugar to the matrix can have an additional ‘cooling’ effect [19]. 4-Hydroxy-3methoxycinnamic acid (ferulic acid) was one of the first matrices for DNA [20]; another matrix studied for MALDI detection of DNA was 2,5-dihydroxybenzoic acid [21]. However, these two matrices are rarely used in practice, due to, among other things, lower sensitivity compared to the “gold standard”, 3-hydroxypicolinic acid, and a tendency to reduce cytosine residues. αCyano-4-hydroxycinnamic acid is a common matrix for peptides, but it can also be used for small DNA products of about five nucleobases; it is significantly less suitable for larger fragments. Other, more rarely used matrices are listed in Table 1 for completeness. More details about properties and handling of these matrices can be found in package inserts of chemical or MS companies. 1.3. Chemical modification of DNA Chemical modification of oligonucleotides is another strategy for improving MALDI detection. This approach was motivated by the observation that thymidine homopolymers are significantly more stable as gas-phase ions than other homopolymers or random sequence oligonucleotides. Moreover, oligonucleotides containing methyl and bromocytidines show improved ion stability [22]. Replacing purines by 7-deaza-analogs is one approach to prevent depurination of DNA [23,24]. A second
Table 1 The typical matrix for UV-MALDI is an aromatic acid, which efficiently absorbs laser energy Matrix
MALDI performance
Comments
3-Hydroxypicolinic acid (3-HPA)
+++
Picolinic acid 6-Aza-2-thiothymine 4-Hydroxy-3-methoxycinnamic acid (ferulic acid) 2,5-Dihydroxybenzoic acid (DHB) 2,4,6-Trihydroxyacetophenone (THAP)
++ ++ + + +++
2,3,4-Trihydroxyacetophenone (THAP)
+++
Alpha-cyano-4-hydroxycinnamic Acid
(++)
Alpha-cyano-4-hydroxycinnamic acid methyl ester Anthranilic acid Salicylamide
(+++) + +
Gold standard for oligonucleotides greater than 10 bases, less suitable for smaller oligonucleotides due to matrix adducts but still good performance Usually a co-matrix of 3-hydroxypicolinic acid Little fragmentation, rarely used in practice Rarely used in practice, rather for oligonucleotides larger than 3500 Mr Rarely used for oligonucleotides An alternative to 3-HPA, particularly for small oligonucleotides, can be mixed with 2,3,4-THAP An alternative to 3-HPA, particularly for small oligonucleotides, can be mixed with 2,4,6-THAP Particularly for PNA, modified and small oligonucleotides with charge-neutral backbones For PNA and modified oligonucleotides with charge-neutral backbones Rarely used in practice, rather for oligonucleotides larger than 3500 Mr Rarely used in practice, rather for oligonucleotides larger than 3500 Mr
“+++” means excellent, “++” good and “+” medium-quality performance in practice. Brackets indicate a limited use of the matrix. More information can be found in package inserts of companies supplying these products (e.g., http://www.sigmaaldrich.com/img/assets/4242/fl_analytix6_2001_new.pdf or http://www.bdal.de/care).
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procedure consists in the use of ribonucleotides containing 2′-OH groups that stabilize the gas-phase ion [25]. Additionally, it was observed that an arabinose modification or a 2′-fluoro modification of the ribose improved oligonucleotide stability [26]. These results have clearly shown that both, the nucleobase and the sugar, affect nucleic acid stability in MALDI. A further concept, termed DNA charge tagging, has focused on the chemical difference of oligonucleotides and peptides [27]. While most peptides are formally uncharged, DNA carries as many negative charges as phosphates. Charges can be neutralized by replacing phosphates by phosphorothioate groups and methylating them. Moreover, the addition of a positively charged tag to these modified oligonucleotides was carried out [28]. The objective of this was to generate a product with a defined charge state, thus relying on the matrix for desorption, but not for ionization. α-Cyano-4-hydroxy-cinnamic acid methyl ester turned out to be a suitable matrix for these DNA products, carrying one positive fixed charge [28]. Using this approach, a hundred-fold increase in detection sensitivity was obtained, similar to peptide analysis. Corresponding results were obtained when all but one phosphate linkage was neutralized and the oligonucleotides contained single, negative, fixed charges [28]. α-Cyano-4-hydroxy-cinnamic acid methyl ester is a derivative of α-cyano-4-hydroxy-cinnamic acid, the most commonly applied matrix for peptide analysis [29]. One of the most striking observations with this matrix is that native DNA cannot be efficiently detected [28]. In contrast to other matrices, it has a significantly higher pKa of around 8 so that this matrix does not support protonation of the oligonucleotides during sample preparation. Its absorption maximum matches the emission wavelength of a nitrogen laser (337 nm). Standard DNA matrices, such as 3-hydroxypicolinic acid, have slightly acidic pKa values of around 4. MALDI analysis of DNA with the use of 3-hydroxypicolinic acid can be performed in the positiveor negative-ion detection mode of the mass spectrometer. The selectivity of the α-cyano-4-hydroxy-cinnamic acid methyl ester, however, is towards singly charged DNA products. The reduction in size of nucleic acids, for example of extension products of primers, is another method for providing higher detection accuracy. This can be achieved by using exonucleases that stop digestion at modified phosphodiester linkages [30]. Another approach is to use chemically cleavable nucleobases [31]. Recently, photocleavable linkers have also been applied. This could be the most straightforward approach, because cleavage takes place in a relatively short time (seconds), and no enzymes or chemical reagents are required [32,33]. However, all these methods imply modification of oligonucleotides, which could interfere with hybridization to target sequences as well as reduce enzymatic reaction efficiencies. Although this article has so far focussed on DNA, the desorption and ionization properties of related nucleic acids, like RNA and other nucleic acids, are generally similar to those of DNA. RNA and oligonucleotides containing charge-neutral backbones might be easier to detect, as desorption/ionization is more efficient while fragmentation is generally less prominent. Moreover, salts are less likely to stick to these molecules than natural DNA. The optimization of the detection of new analytes
consists in identifying a good matrix, including co-matrices and sugars, that will improve analysis, and in finding a suitable preparation method. In practice, 3-hydroxypicolinic will usually turn out to be the most suitable matrix for the majority of nucleic acids. However, it seems that peptide-like analytes, such as peptide-nucleic acids (PNAs) [34] and charge-tagged DNA [28,35], can be more efficiently detected with α-cyano-4hydroxy-cinnamic acid (methyl ester), particularly in the presence of salt. 1.4. Preparation methods There are two common matrix preparation methods, thin-layer and dried-droplet preparation (Fig. 2). Virtually all known MALDI matrices can be applied in dried-droplet procedures, whereas matrices soluble in organic solvents are also useful for thin-layer preparations. For thin-layer preparations, low-microliter volumes of a matrix, like α-cyano-4-hydroxy-cinnamic acid (methyl ester), are applied to the MALDI target plate in a volatile solvent, such as acetone. This can be done by spotting the matrix with a pipette or a liquid-handling robot. Alternatively, the matrix can be sprayed onto a MALDI target plate. The solvent spreads and evaporates immediately, leaving a thin-layer of small matrix crystals. The analyte is dispensed onto the thin-layer in a solvent that does not completely dissolve the matrix, i.e. the sample contains a significant amount of water (40% acetonitrile is a typical solvent). Analyte molecules are built into the surface of the matrix. Alternatively, analyte samples are first deposited on the surface and matrix is subsequently applied. In general, this leads to reduced detection sensitivity and resolution. For dried-droplet preparations, a matrix solution (e.g., 3hydroxypicolinic acid) is mixed with an analyte solution and then spotted onto the MALDI target plate. Alternatively, matrix can be spotted onto the surface. Then, analyte samples are added to the dry matrix, re-dissolving it; this mixture is then again allowed to dry. As for thin-layer preparation, analyte and matrix could also be applied in reverse order. In general, dried-droplet preparations frequently result in “sweet spots”. Certain positions on the preparation give better results than others because laser fluence threshold for desorption/ionization varies from spot to spot. Laser fluence higher than the threshold value decreases mass resolution because the excess energy transferred to the analyte DNA causes metastable decay [19]. This may be overcome by searching for ‘sweet’ spots or miniaturizing sample preparation (Fig. 3) to nanoliter volumes, resulting in complete matrix-analyte desorption by the laser [36]. Another, possibly successful but rarely used approach is to rapidly dry the preparation in a vacuum chamber, potentially yielding smaller analyte-matrix crystals. Provided that well-purified nucleic acid samples are used, this might lead to increased crystal homogeneity and thus improved spot-to-spot reproducibility. Moreover, the laser fluence can be adjusted appropriately. A ‘‘hump’’ of the signal tracing can be a sign of too high a laser fluence and metastable decay of oligonucleotides. Another reason could be sample heterogeneity. The addition of sugars to the matrix can significantly reduce the transfer of excess laser energy to DNAs [19]. For example, fructose and fucose were
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Fig. 2. Three common preparation procedures are shown. Matrix is depicted by light grey and samples by dark-grey ovals. On the left side, the principle of thin-layer preparation is illustrated. For thin-layer preparation, hydrophobic matrices that can be dissolved in acetone, are most commonly used solvent. In general, 0.1–1 μl matrix and analyte solution are applied to a metal plate. In the middle, a typical dried-droplet procedure is shown. For this preparation method, usually 0.1–1 μl of water-soluble matrices, such as 3-hydroxypicolinc acid and similar volumes of analyte solution are used. 3-hydroxypicolinic acid is a good matrix for detecting DNA but it forms heterogeneous crystals, often at the rim of the spots. For automatic analysis it is important to produce homogeneous spots. To overcome this problem, a miniaturized variation of the dried-droplet method (right hand side) was introduced. The Sequenom Company routinely applies this method on silicon chips with nanodispensers. First, matrix is spotted in low-nano-liter volumes (e.g., 4 nl) onto the chip; subsequently, low-nano-liter volumes of purified DNA samples are spotted onto these devices and allowed to co-crystallise. The chips are clamped in an adapter, suitable for ionization chambers of commercially available MALDI mass spectrometers.
found to be effective matrix additives [19]. When these sugars are used, mass resolution of oligonucleotides remains constant as laser energy increases. However, due to the uneven height of dried-droplet preparations in low-micro-liter volumes, the mass calibration can be unstable. MALDI analysis is commonly performed by determination of the time-of-flight (TOF) of an ion (Fig. 1). Variable height of the matrix preparation results in a shift of the starting position, which affects the time of flight. This can easily lead to a mass variation of a few daltons. Thin-layer preparations give less spot-to-spot variation and better mass accuracy and resolution. They were mainly applied with αcyano-4-hydroxy-cinnamic acid for peptide analysis, while DNA analysis preferably was performed with HPA, using a dried-droplet preparation. Using an α-cyano-4-hydroxy-cinnamic acid methyl ester thin-layer preparation results in a significant improvement of the reproducibility of the sample preparation, particularly in automated set-ups. Miniaturization can alleviate the problems associated with dried-droplet preparations [35]. Sequenom⁎ offers fluid nanodispenser devices for printing nano-liter volumes of DNA products onto so-called SpectroCHIPS™, consisting of silicon dioxide (Fig. 3). These chips are covered with spots of ca. 4 nl dried matrix, essentially composed of 3-hydroxypicolinic acid. In other commercially available methods, pre-structured MALDI targets are applied. For example, Bruker Daltonics⁎⁎
has introduced anchor-chip targets⁎San Diego, USA; http:// www.sequenom.com⁎⁎Bremen, Germany; http://www.bdal. com [37] that were coated with hydrophobic Teflon and an array of up to 1536 hydrophilic spots (called “anchors”). Matrix and analyte droplets are deposited onto these spots, either manually or using liquid-handling robots. After solvent evaporation, the sample is concentrated due to the strongly water-repellent nature of the Teflon surface. The initial matrix concentration must be kept low, because matrix and sample concentration by more than 2 orders of magnitudes can be achieved during crystallization. For example, the detection sensitivity of oligonucleotides is in the range of ca. 1 fmol, while for traditional techniques on flat surfaces this would be hardly achievable. Other MS companies introduced similar approaches later. Relative quantification of oligonucleotides, for example DNA products of SNP alleles in pooled DNA samples, has been accomplished by using MALDI [12]. Since the desorption efficiency of oligonucleotides consisting of different base sequences is similar, resulting mass spectra reflect the corresponding proportion of SNP alleles. Relative quantification of mass signals of allele frequencies in the range from 0.1 to 0.9 can be achieved routinely with a reproducibility of about 2% [38]. Quantification via signal intensities heavily depends on the matrix preparation used. Either miniaturized dried-droplet or
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Fig. 3. A photo of a dried-droplet sample preparation with 3-hydroxypicolinic acid is shown. On the left side the conventional dried-droplet procedure with low-microliter matrix and sample volumes is shown. Crystallization of matrix and samples in these volumes usually leads to highly heterogeneous samples. As shown on the right side, miniaturization of the preparation procedure produces fairly homogeneous spots, facilitating automatic measurement of DNA samples.
thin-layer preparation methods can be applied. Using a nano-liter dried-droplet preparation, the whole matrix–analyte mixture is evaporated by the laser, generally leading to reliable quantification of analytes. For thin-layer preparation, however, a number of positions must be analyzed to produce a representative average mass spectrum. 1.5. Purification procedures Liquid chromatography (LC) can be used to purify samples for MALDI-MS. However, LC is time-consuming, and analytes are diluted during elution. Therefore, LC has rarely been employed for DNA sample preparation prior to MALDI. More popular methods include off-line procedures that can be parallelized. Some of them use sample purification by dialysis, ethanol precipitation, and size-exclusion chromatography, or involve various reversed-phase micro-columns [2,7,39]. Other methods require DNA labelling; for instance, the application of magnetic beads, coated with streptavidin, requires DNA to be biotinylated [7]. Oligonucleotides and DNA polymerase substrates such as dideoxy-ribonucleotides can be biotinylated to isolate DNA products of single-nucleotide polymorphisms, derived from primer extension reactions [40]. A nowadays frequently applied, automatable and cost-efficient approach consists in addition of cation-exchange resins to crude oligonucleotide samples, thereby replacing alkali-metal ions by hydrogen or ammonium ions [41]. However, these resins cannot be used to efficiently purify samples from detergents. Other purification approaches provide the possibility of sample preparation directly on the MALDI target or on a suitable slide by means of surface coatings with nucleic acid-binding properties [42]. These surfaces include films of commercial polymers, thin layers of matrix crystals, self-assembled monolayers, and ultra-thin polymer films (more details are described in Ref. [42]). Both hydrophobic and ionic interactions cause analyte adsorption on these surfaces. Moreover, patterned
surfaces with DNA-binding properties enable concentration and purification of analyte molecules. The important role of these surfaces is to immobilize DNA during the washing step, removing buffer components and contamination, which would interfere with MALDI analysis of the DNA sample. Polyethyleneimine, ethoxylated polyethyleneimine, and polyvinylpyrrolidone are suitable materials [43]. Recently, Kepper et al. [43] have introduced gold microscope slides, which were densely coated with primary amino groups that efficiently bind negatively charged DNA. After the slides had been washed, MALDI matrix was spotted onto the analyte DNAs, and oligonucleotides were analyzed in a conventional MALDI mass spectrometer, using suitable slide adapters. On-target purification methods may introduce problems, such as spreading of sample droplets and potentially low homogeneity of samples; miniaturizing the spread of samples can reduce these problems. However, this method involves the use of nano-liter pipetting devices that require some time for rinsing of tips. Another practical problem consists in the adaptation of common mass spectrometers to the geometry of the prototype plates described. 1.6. Outlook MS not only plays a key role in proteomics, but it also provides solutions for genomic applications [44] and DNA-based diagnostics [2]. A plethora of MALDI sample preparation methods have been developed during the last 15 years and are now mature procedures. However, new, more efficient robotic systems, which allow faster and more accurate deposition of nano-liter droplets of matrix and analyte, could significantly improve MALDI analysis of nucleic acids with regard to spectral quality, reproducibility, and throughput. Moreover, MALDIrelated methods that increase sensitivity and dynamic range would represent a significant advance. In this context, reporter molecule tagging methods have been introduced to overcome these limitations. For example, carbocations such as triarylmethyl
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derivatives can be attached to oligonucleotides and detected at attomole levels by laser desorption ionization, both with and without a matrix [45]. Such molecules might also be detectable in vivo or in crude complex biological mixtures (extracts, cells, tissues) and thus significantly extend the application range of MALDI mass spectrometers. Future developments in MALDIMS may also focus on the construction of new instruments, which would allow accurate and sensitive analysis of large DNA molecules, such as PCR products. Another promising approach with regard to target plates might be the use of “active” surfaces that play the role of matrices. For example, porous silicon has been introduced for detecting biological molecules without common matrices in the DIOS procedure (desorption/ionization on silicon) [46]. Problems in this method still consist in potential contamination of the support during fabrication and use in the laboratory and in the limited mass range of detection; it seems that presently only molecules up to ca. 2000 Mr can be efficiently analyzed with this technique. 2. Note added in proof A new matrix, 3,4-diaminobenzophenone (DABP), was recently introduced for MALDI detection [47]. The authors claim that with this matrix oligonucleotides can be analyzed with lower laser powers, resulting in improved detection limits, reduced fragmentation and fewer alkali metal ion adducts compared to common matrices such as 3-hydroxypicolinic acid. However, it seems questionable if this new matrix is useful for routine genomic applications such as SNP genotyping. In our experience, using purified oligonucleotides, the sensitivity of conventional 3-hydroxypicolinic acid is about 50–100-fold higher than with DABP. 50 fmol oligonucleotide seems to be the sensitivity limit of the DABP matrix. In general, in genotyping experiments about 1 pmol products (usually in about 10 μl) are generated leading to concentrations of about 100 fmol/μl. Of these solutions about 0.1–1 μl (10–100 fmol) products are applied onto the MALDI target plate. In a nutshell, this new matrix might only be useful for detection of crude oligonucleotides in high concentrations, for example for quality control of non-purified samples, but is less suitable for robust SNP genotyping. Acknowledgements I would like to acknowledge the European Union (grant LSHG-CT-2004-503155), the German Ministry for Research and Education (NGFN2, grant 01GR0414), and the Max–Planck Society for support. I would also like to thank Tabea Binger for assistance and Peter Riege (Sequenom) for providing Fig. 3. References [1] Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10000 daltons. Anal Chem 1988;60:2299–303. [2] Sauer S. Typing of single nucleotide polymorphisms by MALDI mass spectrometry: principles and diagnostic applications. Clin Chim Acta 2006;363:95–105.
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[24] Kirpekar F, Nordhoff E, Kristiansen K, Roepstorff P, Hahner S, Hillenkamp F. 7-Deaza purine bases offer a higher ion stability in the analysis of DNA by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 1995;9:525–31. [25] Nordhoff E, Cramer R, Karas M, Hillenkamp F, Kirpekar F, Kristiansen K, et al. Ion stability of nucleic acids in infrared matrix-assisted laser desorption/ ionization mass spectrometry. Nucleic Acids Res 1993;21:3347–57. [26] Ono T, Scalf M, Smith LM. 2'-Fluoro modified nucleic acids: polymerasedirected synthesis, properties and stability to analysis by matrix-assisted laser desorption/ionization mass spectrometry. Nucleic Acids Res 1997;25:4581–8. [27] Gut IG, Jeffery WA, Pappin DJC, Beck S. Analysis of DNA by ‘charge tagging’ and matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 1997;11:43–50. [28] Sauer S, Gut IG. Extension of the GOOD assay for genotyping single nucleotide polymorphisms by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 2003;17:1265–72. [29] Gobom J, Schuerenberg M, Mueller M, Theiss D, Lehrach H, Nordhoff E. Alpha-cyano-4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics. Anal Chem 2001;73:434–8. [30] Sauer S, Lechner D, Berlin K, Lehrach H, Escary JL, Fox N, et al. A novel procedure for efficient genotyping of single nucleotide polymorphisms. Nucleic Acids Res 2000;28:e13. [31] Li J, Butler JM, Tan Y, Lin H, Royer S, Ohler L, et al. Single nucleotide polymorphism determination using primer extension and time-of-flight mass spectrometry. Electrophoresis 1999;20:1258–65. [32] Wenzel T, Elssner T, Fahr K, Bimmler J, Richter S, Thomas I, et al. Genosnip: SNP genotyping by MALDI-TOF MS using photocleavable oligonucleotides. Nucleosides Nucleotides Nucleic Acids 2003;22:1579–81. [33] Sauer S, Lehrach H, Reinhardt R. MALDI mass spectrometry analysis of single nucleotide polymorphisms by photocleavage and charge-tagging. Nucleic Acids Res 2003;31:e63. [34] Bauer O, Guerasimova A, Sauer S, Thamm S, Steinfath M, Herwig R, et al. Multiplexed hybridizations of positively charge-tagged peptide nucleic acids detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 2004;18:1821–9. [35] Sauer S, Gelfand DH, Boussicault F, Bauer K, Reichert F, Gut IG. Facile method for automated genotyping of single nucleotide polymorphisms by mass spectrometry. Nucleic Acids Res 2002;30:e22. [36] Little DP, Cornish TJ, O'Donnell MJ, Braun A, Cotter RJ, Koster H, et al. MALDI on a chip: analysis of low-to subfemtomole quantities of synthetic
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Review
The essence of DNA sample preparation for MALDI mass spectrometry Sascha Sauer ⁎ Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics (Prof. H. Lehrach), Ihnestrasse 73, D-14195 Berlin, Germany Received 26 May 2006; accepted 16 October 2006
Abstract Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important analytical technique in nucleic acid research. MALDI is used for quality control of oligonucleotides as well as for analyzing DNA markers. Sample preparation of nucleic acids is crucial for obtaining high-quality mass spectra. Sample purity, solvent content, suitable matrices, and substrate surfaces, as well as laboratory conditions affect spectra quality. This review presents essential information with regard to sample preparation, DNA modification chemistry, and DNA purification, along with a discussion of instrumental advances, which facilitate and extend the applicability of MALDI in genomics. © 2006 Elsevier B.V. All rights reserved. Keywords: MALDI; DNA; Oligonucleotide; Matrix; Purification; SNP
Contents 1.
Introduction . . . . . . . . . . . 1.1. Instrumental aspects . . . 1.2. Matrices . . . . . . . . . 1.3. Chemical modification of 1.4. Preparation methods . . . 1.5. Purification procedures . 1.6. Outlook . . . . . . . . . 2. Note added in proof . . . . . . Acknowledgements . . . . . . . . . References . . . . . . . . . . . . . .
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1. Introduction Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has become a powerful tool in life science (Fig. 1) [1]. MS detects the mass-to-charge ratio of analyte ions either in positive- or in negative-ion mode thus, no labelling and signal clustering, as necessary for fluorescence detection, are required [2]. In MALDI, co-crystals formed by small and acidic organic molecules, termed the “matrix”, and biological com⁎ Tel.: +49 30 84131565; fax: +49 30 84131365. E-mail address: [email protected]. 0165-022X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2006.10.007
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pounds, such as peptides or nucleic acids, are irradiated with a pulsed laser at a wavelength close to a resonant absorption band of the matrix molecules. This induces an energy transfer and desorption process, evaporating analyte and matrix molecules into the gas phase, which is a high vacuum. During MALDI a proton-transfer reaction of matrix and analyte molecules in the gas phase is induced, predominantly leading to singly charged ions [3]. The ions are accelerated by an electric field and guided by ion optics into a mass analyzer. MALDI-MS can be efficiently applied in quality control of oligonucleotides, analyzing markers such as single nucleotide polymorphisms (SNPs) and cytosine methylation, as well as for
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Fig. 1. The principle of MALDI-MS is illustrated. Irradiation of co-crystals formed by organic molecules, called the “matrix”, and oligonucleotides with a pulsed ultraviolet (UV-) laser at a wavelength close to a resonant absorption band of the matrix induces an energy transfer and desorption process, evaporating analyte and matrix molecules into the gas phase (vacuum: ca. 10− 7 mbar). Common UV-lasers are either nitrogen lasers that emit at 337 nm or frequency-tripled Nd:YAG lasers emitting at 355 nm. The photon energy of these UV lasers is in the range of about 3.49–3.68 eV, and the photon width is about 1–5 ns. During the desorption process and/or in the gas phase, ionization of matrix and analytes takes place by proton-transfer reactions. Product ions are accelerated by an electric field. In general, MALDIMS is executed with time-of-flight (TOF) separation: ions are guided by ion optics into a field-free flight-tube (vacuum: ca. 10− 9 mbar) and separated according to their mass-to-charge ratios before they impinge on a micro-channel plate detector (MCP). The scale of this figure is not proportional to the instrumental set-up of common MALDI mass spectrometers.
analysis of allele-specific expression and detection of alternative splicing [4–6]. Most of these approaches involve buffered solvents and a certain amount of salts in reaction solutions. Sample preparation of DNA is important for obtaining highquality MALDI mass spectra [7]. Sample purity, solvent content, substrate surface and laboratory conditions, such as humidity and temperature, affect matrix/analyte co-crystallization. Nucleic acids are harder to detect by MALDI than peptides. Size-dependent fragmentation of DNA during MALDI is a common phenomenon caused by excess energy during laser desorption, which results in a loss of signal intensity for intact DNA. Fragmentation can be induced by protonation of DNA bases, leading to base loss (mainly depurination) [8]. Interestingly, RNAs are more stable in MALDI due to the additional 2′hydroxyl group, which stabilizes the glycosidic bond, leading to significantly reduced depurination and fragmentation of the whole oligomer [9]. An additional limiting factor may be a general bias of MALDI towards smaller DNAs [10]. The sensitivity of commercially available MALDI mass spectrometers is presently in the range of ca. 1 fmol–100 amol per analyte. Modern MALDI mass spectrometers generally use delayed extraction [11], which enables high resolution of mass signals, allowing the distinction of oligonucleotides in the mass range of 1000–10,000 Mr. These instruments are capable of recording at least one spectrum per second, and multiple nucleic acids can be detected in a single measurement (multiplex analysis). Additionally, relative quantification of different nucleic acids is feasible [12]. This can be attributed to the chemical nature of nucleic acids, which, in contrast to peptides, are rather monotonous polymers, practically displaying no differences during laser desorption. The biggest problem in the detection of nucleic acids by MALDI consists in their negatively charged sugar-phosphate backbones [7]. Phosphate residues provide a site of negative charge in solution. Each DNA oligomer carries as many negative charges as phosphate linkages. The affinity of the phosphates for
alkali counterions, such as sodium and potassium, is high, but does not result in complete saturation. Thus these ions interfere with the ionization process, by inducing adducts, and this severely limits signal intensity and quality. The use of ammonium salts in MALDI is a well-known approach to counteract ion affinities [13]. In solution, ammonium exits as an ammonium ion, whereas in the gas phase ammonium is readily lost, resulting in a reduced adduct structure of nucleic acids. However, ammonium ions introduce a degree of suppression to the desorption process, which decreases analytical resolution and sensitivity. Furthermore, residual amounts of denaturants, such as urea or guanidineHCl and detergents, such as Tween-20 or Triton X, interfere with matrix crystallization and decrease or even eliminate signals in MALDI-MS. Purification methods must be applied prior to MALDI to solve the above-mentioned problems with impurities, which are often associated with biological assays. 1.1. Instrumental aspects UV-laser MALDI-MS (Fig. 1) has not only been used for the analysis of oligonucleotides or primer extension products of SNPs with a size of about 5000–8000 Mr, but also for complex mixtures containing large DNA fragments [7] such as DNA sequencing ladders. In general, these products are routinely detected in gene laboratories by capillary electrophoresis [14]. The difficulty of measuring many DNA fragments consists in the amount of ion current available to simultaneously detect different oligonucleotides and the effects this implies on the detection sensitivity and resolution of these analytes. Moreover, due to the abovementioned loss of signal intensity and mass resolution with increasing size of nucleic acids this mass spectrometric technology is limited to nucleic acids smaller than ca. 100 nucleotides. Masses as small as 9 Mr — which is the mass difference between thymine and adenine — are virtually impossible to resolve at ca. 30,000 Mr by conventional UV-MALDI. Hillenkamp and coworkers [15] have introduced infrared lasers, such as ER:YAG
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lasers, emitting at 2.94 μm with a pulse width at 85 ns, for detection of large nucleic acids, using glycerol as matrix. One problem in the MALDI analysis of large biological molecules is the sensitivity of the micro-channel plate detectors commonly used in most MALDI mass spectrometers, which drops down with increasing particle mass. The peak-width of large DNA fragments of about several hundred bases, such as PCR products, can range over several 100 Mr, and the mass accuracy is quite poor. Cryogenic detectors might be a solution to overcome this problem, but these instruments are presently far from mature enough for routine application [15]. Moreover, inherently complex signals of macromolecules result from atomic isotopes; in the case of DNA this problem could be counteracted by using expensive isotope-free nucleotides. 1.2. Matrices Matrices for MALDI are applied on evenly flat conductive plates that are introduced in the ionization chamber of the mass spectrometer. Stainless steel, aluminum/nickel alloys, and gold, or semi-conductors, such as silicon, are most commonly used plate materials. In general, the optimization of MALDI depends on identifying the best matrix and preparation method for a particular analyte. The mechanism of MALDI is not well understood and the interaction of DNA with a matrix during the desorption/ionization process eludes further investigation. However, empirical findings have aided the method. MALDI sample preparation is generally performed with acidic matrices, and acidic conditions are encountered during desorption and ionization. MALDI matrices must meet a number of requirements: (1) they must be able to embed analyte molecules, (2) they must be soluble in the same solvents as the analytes, (3) they have to be stable in vacuum, (4) they should induce co-desorption of analytes, and (5) they should promote ionization of analytes. In general, the matrix-to-analyte mixtures are in the range from 1000:1 to 10,000:1, but this can vary, depending on the preparation. Common matrices for the detection of nucleic acids are listed in Table 1.
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As mentioned above, fragmentation of DNA, frequently observed in MALDI, results from excess energy, deposited during laser irradiation. A detectable degree of depurination has been observed for large DNAs. Fragmentation mainly depends on the nature of the matrix used. A remarkable dependence of the matrix-to-DNA ratio on fragmentation was observed [8]. MALDI matrices, such as 2,3,4- or 2,4,6-trihydroxyacetophenone [16] are termed ‘hot’ if they result in excessive fragmentation, and therefore they are suitable only for small oligonucleotides. Comparatively ‘cool’ matrices, such as 3hydroxypicolinic acid [17] and picolinic acid, as well as 6-aza-2thiothymine [18], result in very little fragmentation and are also suitable for larger nucleic acids. Addition of a sugar to the matrix can have an additional ‘cooling’ effect [19]. 4-Hydroxy-3methoxycinnamic acid (ferulic acid) was one of the first matrices for DNA [20]; another matrix studied for MALDI detection of DNA was 2,5-dihydroxybenzoic acid [21]. However, these two matrices are rarely used in practice, due to, among other things, lower sensitivity compared to the “gold standard”, 3-hydroxypicolinic acid, and a tendency to reduce cytosine residues. αCyano-4-hydroxycinnamic acid is a common matrix for peptides, but it can also be used for small DNA products of about five nucleobases; it is significantly less suitable for larger fragments. Other, more rarely used matrices are listed in Table 1 for completeness. More details about properties and handling of these matrices can be found in package inserts of chemical or MS companies. 1.3. Chemical modification of DNA Chemical modification of oligonucleotides is another strategy for improving MALDI detection. This approach was motivated by the observation that thymidine homopolymers are significantly more stable as gas-phase ions than other homopolymers or random sequence oligonucleotides. Moreover, oligonucleotides containing methyl and bromocytidines show improved ion stability [22]. Replacing purines by 7-deaza-analogs is one approach to prevent depurination of DNA [23,24]. A second
Table 1 The typical matrix for UV-MALDI is an aromatic acid, which efficiently absorbs laser energy Matrix
MALDI performance
Comments
3-Hydroxypicolinic acid (3-HPA)
+++
Picolinic acid 6-Aza-2-thiothymine 4-Hydroxy-3-methoxycinnamic acid (ferulic acid) 2,5-Dihydroxybenzoic acid (DHB) 2,4,6-Trihydroxyacetophenone (THAP)
++ ++ + + +++
2,3,4-Trihydroxyacetophenone (THAP)
+++
Alpha-cyano-4-hydroxycinnamic Acid
(++)
Alpha-cyano-4-hydroxycinnamic acid methyl ester Anthranilic acid Salicylamide
(+++) + +
Gold standard for oligonucleotides greater than 10 bases, less suitable for smaller oligonucleotides due to matrix adducts but still good performance Usually a co-matrix of 3-hydroxypicolinic acid Little fragmentation, rarely used in practice Rarely used in practice, rather for oligonucleotides larger than 3500 Mr Rarely used for oligonucleotides An alternative to 3-HPA, particularly for small oligonucleotides, can be mixed with 2,3,4-THAP An alternative to 3-HPA, particularly for small oligonucleotides, can be mixed with 2,4,6-THAP Particularly for PNA, modified and small oligonucleotides with charge-neutral backbones For PNA and modified oligonucleotides with charge-neutral backbones Rarely used in practice, rather for oligonucleotides larger than 3500 Mr Rarely used in practice, rather for oligonucleotides larger than 3500 Mr
“+++” means excellent, “++” good and “+” medium-quality performance in practice. Brackets indicate a limited use of the matrix. More information can be found in package inserts of companies supplying these products (e.g., http://www.sigmaaldrich.com/img/assets/4242/fl_analytix6_2001_new.pdf or http://www.bdal.de/care).
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procedure consists in the use of ribonucleotides containing 2′-OH groups that stabilize the gas-phase ion [25]. Additionally, it was observed that an arabinose modification or a 2′-fluoro modification of the ribose improved oligonucleotide stability [26]. These results have clearly shown that both, the nucleobase and the sugar, affect nucleic acid stability in MALDI. A further concept, termed DNA charge tagging, has focused on the chemical difference of oligonucleotides and peptides [27]. While most peptides are formally uncharged, DNA carries as many negative charges as phosphates. Charges can be neutralized by replacing phosphates by phosphorothioate groups and methylating them. Moreover, the addition of a positively charged tag to these modified oligonucleotides was carried out [28]. The objective of this was to generate a product with a defined charge state, thus relying on the matrix for desorption, but not for ionization. α-Cyano-4-hydroxy-cinnamic acid methyl ester turned out to be a suitable matrix for these DNA products, carrying one positive fixed charge [28]. Using this approach, a hundred-fold increase in detection sensitivity was obtained, similar to peptide analysis. Corresponding results were obtained when all but one phosphate linkage was neutralized and the oligonucleotides contained single, negative, fixed charges [28]. α-Cyano-4-hydroxy-cinnamic acid methyl ester is a derivative of α-cyano-4-hydroxy-cinnamic acid, the most commonly applied matrix for peptide analysis [29]. One of the most striking observations with this matrix is that native DNA cannot be efficiently detected [28]. In contrast to other matrices, it has a significantly higher pKa of around 8 so that this matrix does not support protonation of the oligonucleotides during sample preparation. Its absorption maximum matches the emission wavelength of a nitrogen laser (337 nm). Standard DNA matrices, such as 3-hydroxypicolinic acid, have slightly acidic pKa values of around 4. MALDI analysis of DNA with the use of 3-hydroxypicolinic acid can be performed in the positiveor negative-ion detection mode of the mass spectrometer. The selectivity of the α-cyano-4-hydroxy-cinnamic acid methyl ester, however, is towards singly charged DNA products. The reduction in size of nucleic acids, for example of extension products of primers, is another method for providing higher detection accuracy. This can be achieved by using exonucleases that stop digestion at modified phosphodiester linkages [30]. Another approach is to use chemically cleavable nucleobases [31]. Recently, photocleavable linkers have also been applied. This could be the most straightforward approach, because cleavage takes place in a relatively short time (seconds), and no enzymes or chemical reagents are required [32,33]. However, all these methods imply modification of oligonucleotides, which could interfere with hybridization to target sequences as well as reduce enzymatic reaction efficiencies. Although this article has so far focussed on DNA, the desorption and ionization properties of related nucleic acids, like RNA and other nucleic acids, are generally similar to those of DNA. RNA and oligonucleotides containing charge-neutral backbones might be easier to detect, as desorption/ionization is more efficient while fragmentation is generally less prominent. Moreover, salts are less likely to stick to these molecules than natural DNA. The optimization of the detection of new analytes
consists in identifying a good matrix, including co-matrices and sugars, that will improve analysis, and in finding a suitable preparation method. In practice, 3-hydroxypicolinic will usually turn out to be the most suitable matrix for the majority of nucleic acids. However, it seems that peptide-like analytes, such as peptide-nucleic acids (PNAs) [34] and charge-tagged DNA [28,35], can be more efficiently detected with α-cyano-4hydroxy-cinnamic acid (methyl ester), particularly in the presence of salt. 1.4. Preparation methods There are two common matrix preparation methods, thin-layer and dried-droplet preparation (Fig. 2). Virtually all known MALDI matrices can be applied in dried-droplet procedures, whereas matrices soluble in organic solvents are also useful for thin-layer preparations. For thin-layer preparations, low-microliter volumes of a matrix, like α-cyano-4-hydroxy-cinnamic acid (methyl ester), are applied to the MALDI target plate in a volatile solvent, such as acetone. This can be done by spotting the matrix with a pipette or a liquid-handling robot. Alternatively, the matrix can be sprayed onto a MALDI target plate. The solvent spreads and evaporates immediately, leaving a thin-layer of small matrix crystals. The analyte is dispensed onto the thin-layer in a solvent that does not completely dissolve the matrix, i.e. the sample contains a significant amount of water (40% acetonitrile is a typical solvent). Analyte molecules are built into the surface of the matrix. Alternatively, analyte samples are first deposited on the surface and matrix is subsequently applied. In general, this leads to reduced detection sensitivity and resolution. For dried-droplet preparations, a matrix solution (e.g., 3hydroxypicolinic acid) is mixed with an analyte solution and then spotted onto the MALDI target plate. Alternatively, matrix can be spotted onto the surface. Then, analyte samples are added to the dry matrix, re-dissolving it; this mixture is then again allowed to dry. As for thin-layer preparation, analyte and matrix could also be applied in reverse order. In general, dried-droplet preparations frequently result in “sweet spots”. Certain positions on the preparation give better results than others because laser fluence threshold for desorption/ionization varies from spot to spot. Laser fluence higher than the threshold value decreases mass resolution because the excess energy transferred to the analyte DNA causes metastable decay [19]. This may be overcome by searching for ‘sweet’ spots or miniaturizing sample preparation (Fig. 3) to nanoliter volumes, resulting in complete matrix-analyte desorption by the laser [36]. Another, possibly successful but rarely used approach is to rapidly dry the preparation in a vacuum chamber, potentially yielding smaller analyte-matrix crystals. Provided that well-purified nucleic acid samples are used, this might lead to increased crystal homogeneity and thus improved spot-to-spot reproducibility. Moreover, the laser fluence can be adjusted appropriately. A ‘‘hump’’ of the signal tracing can be a sign of too high a laser fluence and metastable decay of oligonucleotides. Another reason could be sample heterogeneity. The addition of sugars to the matrix can significantly reduce the transfer of excess laser energy to DNAs [19]. For example, fructose and fucose were
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Fig. 2. Three common preparation procedures are shown. Matrix is depicted by light grey and samples by dark-grey ovals. On the left side, the principle of thin-layer preparation is illustrated. For thin-layer preparation, hydrophobic matrices that can be dissolved in acetone, are most commonly used solvent. In general, 0.1–1 μl matrix and analyte solution are applied to a metal plate. In the middle, a typical dried-droplet procedure is shown. For this preparation method, usually 0.1–1 μl of water-soluble matrices, such as 3-hydroxypicolinc acid and similar volumes of analyte solution are used. 3-hydroxypicolinic acid is a good matrix for detecting DNA but it forms heterogeneous crystals, often at the rim of the spots. For automatic analysis it is important to produce homogeneous spots. To overcome this problem, a miniaturized variation of the dried-droplet method (right hand side) was introduced. The Sequenom Company routinely applies this method on silicon chips with nanodispensers. First, matrix is spotted in low-nano-liter volumes (e.g., 4 nl) onto the chip; subsequently, low-nano-liter volumes of purified DNA samples are spotted onto these devices and allowed to co-crystallise. The chips are clamped in an adapter, suitable for ionization chambers of commercially available MALDI mass spectrometers.
found to be effective matrix additives [19]. When these sugars are used, mass resolution of oligonucleotides remains constant as laser energy increases. However, due to the uneven height of dried-droplet preparations in low-micro-liter volumes, the mass calibration can be unstable. MALDI analysis is commonly performed by determination of the time-of-flight (TOF) of an ion (Fig. 1). Variable height of the matrix preparation results in a shift of the starting position, which affects the time of flight. This can easily lead to a mass variation of a few daltons. Thin-layer preparations give less spot-to-spot variation and better mass accuracy and resolution. They were mainly applied with αcyano-4-hydroxy-cinnamic acid for peptide analysis, while DNA analysis preferably was performed with HPA, using a dried-droplet preparation. Using an α-cyano-4-hydroxy-cinnamic acid methyl ester thin-layer preparation results in a significant improvement of the reproducibility of the sample preparation, particularly in automated set-ups. Miniaturization can alleviate the problems associated with dried-droplet preparations [35]. Sequenom⁎ offers fluid nanodispenser devices for printing nano-liter volumes of DNA products onto so-called SpectroCHIPS™, consisting of silicon dioxide (Fig. 3). These chips are covered with spots of ca. 4 nl dried matrix, essentially composed of 3-hydroxypicolinic acid. In other commercially available methods, pre-structured MALDI targets are applied. For example, Bruker Daltonics⁎⁎
has introduced anchor-chip targets⁎San Diego, USA; http:// www.sequenom.com⁎⁎Bremen, Germany; http://www.bdal. com [37] that were coated with hydrophobic Teflon and an array of up to 1536 hydrophilic spots (called “anchors”). Matrix and analyte droplets are deposited onto these spots, either manually or using liquid-handling robots. After solvent evaporation, the sample is concentrated due to the strongly water-repellent nature of the Teflon surface. The initial matrix concentration must be kept low, because matrix and sample concentration by more than 2 orders of magnitudes can be achieved during crystallization. For example, the detection sensitivity of oligonucleotides is in the range of ca. 1 fmol, while for traditional techniques on flat surfaces this would be hardly achievable. Other MS companies introduced similar approaches later. Relative quantification of oligonucleotides, for example DNA products of SNP alleles in pooled DNA samples, has been accomplished by using MALDI [12]. Since the desorption efficiency of oligonucleotides consisting of different base sequences is similar, resulting mass spectra reflect the corresponding proportion of SNP alleles. Relative quantification of mass signals of allele frequencies in the range from 0.1 to 0.9 can be achieved routinely with a reproducibility of about 2% [38]. Quantification via signal intensities heavily depends on the matrix preparation used. Either miniaturized dried-droplet or
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Fig. 3. A photo of a dried-droplet sample preparation with 3-hydroxypicolinic acid is shown. On the left side the conventional dried-droplet procedure with low-microliter matrix and sample volumes is shown. Crystallization of matrix and samples in these volumes usually leads to highly heterogeneous samples. As shown on the right side, miniaturization of the preparation procedure produces fairly homogeneous spots, facilitating automatic measurement of DNA samples.
thin-layer preparation methods can be applied. Using a nano-liter dried-droplet preparation, the whole matrix–analyte mixture is evaporated by the laser, generally leading to reliable quantification of analytes. For thin-layer preparation, however, a number of positions must be analyzed to produce a representative average mass spectrum. 1.5. Purification procedures Liquid chromatography (LC) can be used to purify samples for MALDI-MS. However, LC is time-consuming, and analytes are diluted during elution. Therefore, LC has rarely been employed for DNA sample preparation prior to MALDI. More popular methods include off-line procedures that can be parallelized. Some of them use sample purification by dialysis, ethanol precipitation, and size-exclusion chromatography, or involve various reversed-phase micro-columns [2,7,39]. Other methods require DNA labelling; for instance, the application of magnetic beads, coated with streptavidin, requires DNA to be biotinylated [7]. Oligonucleotides and DNA polymerase substrates such as dideoxy-ribonucleotides can be biotinylated to isolate DNA products of single-nucleotide polymorphisms, derived from primer extension reactions [40]. A nowadays frequently applied, automatable and cost-efficient approach consists in addition of cation-exchange resins to crude oligonucleotide samples, thereby replacing alkali-metal ions by hydrogen or ammonium ions [41]. However, these resins cannot be used to efficiently purify samples from detergents. Other purification approaches provide the possibility of sample preparation directly on the MALDI target or on a suitable slide by means of surface coatings with nucleic acid-binding properties [42]. These surfaces include films of commercial polymers, thin layers of matrix crystals, self-assembled monolayers, and ultra-thin polymer films (more details are described in Ref. [42]). Both hydrophobic and ionic interactions cause analyte adsorption on these surfaces. Moreover, patterned
surfaces with DNA-binding properties enable concentration and purification of analyte molecules. The important role of these surfaces is to immobilize DNA during the washing step, removing buffer components and contamination, which would interfere with MALDI analysis of the DNA sample. Polyethyleneimine, ethoxylated polyethyleneimine, and polyvinylpyrrolidone are suitable materials [43]. Recently, Kepper et al. [43] have introduced gold microscope slides, which were densely coated with primary amino groups that efficiently bind negatively charged DNA. After the slides had been washed, MALDI matrix was spotted onto the analyte DNAs, and oligonucleotides were analyzed in a conventional MALDI mass spectrometer, using suitable slide adapters. On-target purification methods may introduce problems, such as spreading of sample droplets and potentially low homogeneity of samples; miniaturizing the spread of samples can reduce these problems. However, this method involves the use of nano-liter pipetting devices that require some time for rinsing of tips. Another practical problem consists in the adaptation of common mass spectrometers to the geometry of the prototype plates described. 1.6. Outlook MS not only plays a key role in proteomics, but it also provides solutions for genomic applications [44] and DNA-based diagnostics [2]. A plethora of MALDI sample preparation methods have been developed during the last 15 years and are now mature procedures. However, new, more efficient robotic systems, which allow faster and more accurate deposition of nano-liter droplets of matrix and analyte, could significantly improve MALDI analysis of nucleic acids with regard to spectral quality, reproducibility, and throughput. Moreover, MALDIrelated methods that increase sensitivity and dynamic range would represent a significant advance. In this context, reporter molecule tagging methods have been introduced to overcome these limitations. For example, carbocations such as triarylmethyl
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derivatives can be attached to oligonucleotides and detected at attomole levels by laser desorption ionization, both with and without a matrix [45]. Such molecules might also be detectable in vivo or in crude complex biological mixtures (extracts, cells, tissues) and thus significantly extend the application range of MALDI mass spectrometers. Future developments in MALDIMS may also focus on the construction of new instruments, which would allow accurate and sensitive analysis of large DNA molecules, such as PCR products. Another promising approach with regard to target plates might be the use of “active” surfaces that play the role of matrices. For example, porous silicon has been introduced for detecting biological molecules without common matrices in the DIOS procedure (desorption/ionization on silicon) [46]. Problems in this method still consist in potential contamination of the support during fabrication and use in the laboratory and in the limited mass range of detection; it seems that presently only molecules up to ca. 2000 Mr can be efficiently analyzed with this technique. 2. Note added in proof A new matrix, 3,4-diaminobenzophenone (DABP), was recently introduced for MALDI detection [47]. The authors claim that with this matrix oligonucleotides can be analyzed with lower laser powers, resulting in improved detection limits, reduced fragmentation and fewer alkali metal ion adducts compared to common matrices such as 3-hydroxypicolinic acid. However, it seems questionable if this new matrix is useful for routine genomic applications such as SNP genotyping. In our experience, using purified oligonucleotides, the sensitivity of conventional 3-hydroxypicolinic acid is about 50–100-fold higher than with DABP. 50 fmol oligonucleotide seems to be the sensitivity limit of the DABP matrix. In general, in genotyping experiments about 1 pmol products (usually in about 10 μl) are generated leading to concentrations of about 100 fmol/μl. Of these solutions about 0.1–1 μl (10–100 fmol) products are applied onto the MALDI target plate. In a nutshell, this new matrix might only be useful for detection of crude oligonucleotides in high concentrations, for example for quality control of non-purified samples, but is less suitable for robust SNP genotyping. Acknowledgements I would like to acknowledge the European Union (grant LSHG-CT-2004-503155), the German Ministry for Research and Education (NGFN2, grant 01GR0414), and the Max–Planck Society for support. I would also like to thank Tabea Binger for assistance and Peter Riege (Sequenom) for providing Fig. 3. References [1] Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10000 daltons. Anal Chem 1988;60:2299–303. [2] Sauer S. Typing of single nucleotide polymorphisms by MALDI mass spectrometry: principles and diagnostic applications. Clin Chim Acta 2006;363:95–105.
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