Anal. Chem. 2005, 77, 4999-5008
Detection of DNA Sequence Variations in Homoand Heterozygous Samples via Molecular Mass Measurements by Electrospray Ionization Time-of-Flight Mass Spectrometry Herbert Oberacher,*,† Harald Niedersta 1 tter,† Bruno Casetta,‡ and Walther Parson†
Institute of Legal Medicine, Innsbruck Medical University, Muellerstrasse 44, 6020 Innsbruck, Austria, and Applied Biosystems, Via Tiepolo 18, I-20052 Monza (MI), Italy
The potential of ion-pair reversed-phase high-performance liquid chromatography on-line hyphenated to electrospray ionization time-of-flight mass spectrometry for the characterization of polymerase chain reaction (PCR) amplified nucleic acids was evaluated. For that purpose, a “SNP toolbox” was constructed by cloning and PCR-mediated site-directed in vitro mutagenesis at nucleotide position (ntp) 16 519 of a sequence-verified fragment of the human mitochondrial genome (ntps 15 900-599). Confirmatory sequencing demonstrated that within the sequences of the clones one and the same base was mutated to all other bases. Using these clones or equimolar mixtures of these clones as PCR templates, 51-401-bplong amplicons were generated, which were used to determine the upper size limits of PCR products for the unequivocal detection of sequence variations in homoand heterozygous samples. Based on the high mass spectrometric performance of the applied time-of-flight mass spectrometer, the unequivocal genotyping of all kinds of single base exchanges in PCR amplicons from heterozygous samples with lengths up to 254 base pairs (bp) was demonstrated. Considering homozygous samples, the successful genotyping of single base substitutions in up to 401-bp-long PCR products was possible. Consequently, the described hyphenated technique represents one of the most powerful mass spectrometric genotyping assays available today. The frequency of single nucleotide polymorphisms (SNPs) across the genome is known to be greater than that of any other type of genetic marker,1,2 which gives them definite utility for trait or disease gene discovery purposes, evolution genetic studies, and forensic casework. Accordingly, the genotyping of SNPs has gained more and more attraction over the last years. Many different assays based on several different detection principles have been introduced and reviewed in detail.3-12 In nearly all * To whom correspondence should be addressed. Tel.: +43 512 507 3322. Fax: +43 512 507 2764. E-mail:
[email protected]. † Innsbruck Medical University. ‡ Applied Biosystems. (1) Cooper, D. N.; Krawczak, M. Human gene mutation; Academic Press: New York, 1999. (2) Brookes, A. J. Gene 1999, 234, 177-86. 10.1021/ac050399f CCC: $30.25 Published on Web 06/21/2005
© 2005 American Chemical Society
common techniques, the allelic state is determined by measuring values such as migration times, retention times, and hybridization amounts, which are indirectly related to the present allelic state. As these terms are easily influenced by the physical environment, miscalling is a common concern. By comparison, mass spectrometry (MS) is based on the measurement of an intrinsic property of a nucleic acidsits molecular masssand is therefore more accurate than any other genotyping approach. MS of nucleic acids has been triggered by the introduction of electrospray ionization (ESI)13 and matrix-assisted laser desorption/ionization (MALDI)14 as soft ionization techniques. The first publications describing DNA analysis by MALDI appeared in the early 1990s.15-17 However, even after more than 10 years of research, the inherent problems of MALDI, such as adduct formation with alkali ions as well as fragmentation of labile glycosidic bonds, have not been solved yet. So far, it is not possible to resolve an A/T exchange showing the smallest mass difference of only 9.01 atomic mass units (amu) in nucleic acids larger than ∼30 nucleotides (nt).18,19 Consequently, MALDI cannot be applied to the direct genotyping of polymerase chain reaction (PCR) products. A number of different strategies have been developed to shorten the allele-specific product to a length, which is (3) Cotton, R. G. Trends Genet. 1997, 13, 43-6. (4) Nollau, P.; Wagener, C. Clin. Chem. 1997, 43, 1114-28. (5) Syvanen, A. C. Hum. Mutat. 1999, 13, 1-10. (6) Mir, K. U.; Southern, E. M. Annu. Rev. Genomics Hum. Genet. 2000, 1, 329-60. (7) Gut, I. G. Hum. Mutat. 2001, 17, 475-92. (8) Brennan, M. D. Am. J. Pharmacogenomics 2001, 1, 295-302. (9) Kwok, P. Y. Annu. Rev. Genomics Hum. Genet. 2001, 2, 235-58. (10) Kristensen, V. N.; Kelefiotis, D.; Kristensen, T.; Borresen-Dale, A. L. Biotechniques 2001, 30, 318-22. (11) Syvanen, A. C. Nat. Rev. Genet. 2001, 2, 930-42. (12) Koch, W. H. Nat. Rev. Drug Discovery 2004, 3, 749-61. (13) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985, 57, 675-9. (14) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-301. (15) Huth-Fehre, T.; Gosine, J. N.; Wu, K. J.; Becker, C. H. Rapid Commun. Mass Spectrom. 1992, 6, 209-13. (16) Wu, K. J.; Steding, A.; Becker, C. H. Rapid Commun. Mass Spectrom. 1993, 7, 142-6. (17) Nordhoff, E.; Kirpekar, F.; Karas, M.; Cramer, R.; Hahner, S.; Hillenkamp, F.; Kristiansen, K.; Roepstroff, P.; Lezius, A. Nucleic Acids Res. 1994, 22, 2460-5. (18) Fei, Z.; Ono, T.; Smith, L. M. Nucleic Acids Res. 1998, 26, 2827-8. (19) Shahgholi, M.; Garcia, B. A.; Chiu, N. H.; Heaney, P. J.; Tang, K. Nucleic Acids Res. 2001, 29, E91.
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compatible with the technical limitations of MALDI. Several reviews have summarized the reaction principles, which include hybridization, ligation, cleavage, and primer extension.20-29 Thus, elaborate sample preparation and high reagent costs are common disadvantages of MALDI-based techniques. Regardless of technical limitations, MALDI has found broad applicability for the genotyping of SNPs. ESI represents the alternative technique for the mass spectrometric analysis of nucleic acids, which is known to have a gentler ionization process than MALDI. However, the advantageous feature of ESIsthe transfer of intact, double-stranded PCR products from solution into the gas phasesis disadvantageous in the context of SNP genotyping. Base substitutions are difficult to identify in double-helical DNA by molecular mass measurements, because A-T and G-C base pairs have very similar average masses of 617.4 and 618.4 amu, respectively, and A/T or G/C substitutions do not cause any mass shift at all. Denaturation of double-stranded PCR products into the corresponding single strands prior to their mass spectrometric characterization is advantageous because of the division in half of the masses of the detected species and the possibility to identify base substitutions by measuring mass differences ranging in size between 9.01 and 40.02 amu. Commonly applied denaturing techniques include thermal denaturation with a resistively heated ESI source,30 affinity solid-phase extraction of biotin-labeled single strands with magnetic streptavidin particles,31 or selective digestion with λ exonuclease of one strand labeled with a 5′-terminal phosphate group.32,33 However, these methods are usually laborious and add to the costs and complexity of sample preparation, which probably explains the so far restricted use of ESI-MS for SNP genotyping. Liquid chromatography was introduced as a highly efficient sample preparation technique for the purification and separation of nucleic acids prior to ESI-MS.34,35 As separations are performed at elevated temperatures (65-80 °C), chromatography represents an elegant way for denaturing double-stranded nucleic acids into the corresponding single strands, simplifying mass spectrometric genotyping. Hence, the hyphenation of ion-pair reversed-phase high-performance liquid chromatography and ESI-MS (ICEMS) (20) Graber, J. H.; Smith, C. L.; Cantor, C. R. Genet. Anal. 1999, 14, 215-9. (21) Griffin, T. J.; Smith, L. M. Trends Biotechnol. 2000, 18, 77-84. (22) Tost, J.; Gut, I. G. Mass Spectrom. Rev. 2002, 21, 388-418. (23) Pusch, W.; Wurmbach, J. H.; Thiele, H.; Kostrzewa, M. Pharmacogenomics 2002, 3, 537-48. (24) Sauer, S.; Gut, I. G. J. Chromatogr., B 2002, 782, 73-7. (25) Marvin, L. F.; Roberts, M. A.; Fay, L. B. Clin. Chim. Acta 2003, 337, 1121. (26) Jurinke, C.; Oeth, P.; van den Boom, D. Mol. Biotechnol. 2004, 26, 14764. (27) Meng, Z.; Simmons-Willis, T. A.; Limbach, P. A. Biomol. Eng. 2004, 21, 1-13. (28) Tang, K.; Oeth, P.; Kammerer, S.; Denissenko, M. F.; Ekblom, J.; Jurinke, C.; van den Boom, D.; Braun, A.; Cantor, C. R. J. Proteome Res. 2004, 3, 218-27. (29) Gut, I. G. Hum. Mutat. 2004, 23, 437-41. (30) Mangrum, J. B.; Flora, J. W.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2002, 13, 232-40. (31) Krahmer, M. T.; Johnson, Y. A.; Walters, J. J.; Fox, K. F.; Fox, A.; Nagpal, M. Anal. Chem. 1999, 71, 2893-900. (32) Null, A. P.; Hannis, J. C.; Muddiman, D. C. Analyst 2000, 125, 619-26. (33) Null, A. P.; Hannis, J. C.; Muddiman, D. C. Anal. Chem. 2001, 73, 451421. (34) Premstaller, A.; Oberacher, H.; Huber, C. G. Anal. Chem. 2000, 72, 438693. (35) Oberacher, H.; Huber, C. G. TrAC, Trends Anal. Chem. 2002, 21, 166-74.
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proved to be an efficient method for the detection of variations within PCR-amplified sequences.36-42 Due to the limited efficiency of the applied quadrupole ion trap mass analyzer, the maximum allowable amplicon length was limited to ∼70 base pairs (bp) to secure the unequivocal detection of all kind of single base exchanges.41 Consequently, the combination of ICEMS with a time-of-flight (TOF) mass spectrometer offering a comparably higher mass spectrometric performance was suggested. In this report, the potential of the proposed hyphenated technique was evaluated by analyzing amplicons obtained from homo- and heterozygous samples with lengths between 51 and 401 bp. Based on the high mass spectrometric performance of the TOF mass analyzer, the detection of all kinds of single base exchanges was possible in PCR amplicons from heterozygous samples with lengths up to 254 bp. Moreover, the successful genotyping of polymorphisms in up to 401-bp-long PCR products obtained from homozygous samples was demonstrated. In the established configuration, ICEMS represents one of the most powerful mass spectrometric genotyping tools available today. EXPERIMENTAL SECTION Chemicals and Oligonucleotides. Acetonitrile (HPLC gradient grade) was obtained from Merck (Darmstadt, Germany). A stock solution (0.50 M) of butyldimethylammonium bicarbonate (BDMAB) was prepared by passing carbon dioxide gas (Air Liquide, Schwechat, Austria) through a 0.50 M aqueous solution of butyldimethylamine (Fluka, Buchs, Switzerland) at 5 °C until pH 8.4 was reached. A stock solution (0.50 M) of butyldimethylammonium acetate (BDMAA) was prepared by titration of a 0.5 M solution of butyldimethylamine with acetic acid (Fluka) at 5 °C until pH 8.3 was reached. For preparation of all solutions, HPLC-grade water (Merck) was used. All oligonucleotides were obtained from Eurogentec (Seraing, Belgium). Sequences of primers are summarized in Table 1. PCR-Mediated Site-Directed in Vitro Mutagenesis. PCRmediated site-directed in vitro mutagenesis was performed by overlap extension43,44 using a plasmid containing a sequence-verified human mitochondrial control region (nucleotide positions (ntps) 15 900-599) harboring the 16519T allele as template (p16519T). In a first round of PCR (SOE1), two fragments with a 33-bplong sequence overlap containing the intended base substitution at ntp 16 519 were amplified for each substitution in two separate reactions with the primer pairs p1F-16519C/A/G 5′SOE R and 16519C/A/G 3′SOE F-p1272R (Table 1). PCR was conducted in a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, (36) Huber, C. G.; Oberacher, H. Mass Spectrom. Rev. 2001, 310-43. (37) Oberacher, H.; Oefner, P. J.; Parson, W.; Huber, C. G. Angew. Chem., Int. Ed. 2001, 40, 3828-30. (38) Oberacher, H.; Parson, W.; Mu ¨ hlmann, R.; Huber, C. G. Anal. Chem. 2001, 73, 5109-15. (39) Oberacher, H.; Oefner, P. J.; Ho¨lzl, G.; Premstaller, A.; Huber, C. G. Nucleic Acids Res. 2002, 30, e67. (40) Berger, B.; Ho ¨lzl, G.; Oberacher, H.; Niedersta¨tter, H.; Huber, C. G.; Parson, W. J Chromatogr., B 2002, 782, 89-97. (41) Oberacher, H.; Huber, C. G.; Oefner, P. J. Hum. Mutat. 2003, 21, 86-95. (42) Oberacher, H.; Parson, W.; Holzl, G.; Oefner, P. J.; Huber, C. G. J. Am. Soc. Mass Spectrom. 2004, 15, 1897-906. (43) Higuchi, R.; Krummel, B.; Saiki, R. K. Nucleic Acids Res. 1988, 16, 735167. (44) Ho, S. N.; Hunt, H. D.; Horton, R. M.; Pullen, J. K.; Pease, L. R. Gene 1989, 77, 51-9.
Table 1. Primers and Primer Combinations Used in This Study
16519C 3′SOE F: 16519C 5′SOE R: 16519A 3′SOE F: 16519A 5′SOE R: 16519G 3′SOE F: 16519G 5′SOE R: p1F: p298F: p391F: p498F: p594F: p644R: p698R: p1272R: amplicon length (bp)
(a) PCR-Mediated Site-Directed in Vitro Mutagenesis 5′-GGTTCCTACTTCAGGGCCATAAAGCCTAAATAG-3′ 5′-CTATTTAGGCTTTATGGCCCTGAAGTAGGAACC-3′ 5′-GGTTCCTACTTCAGGGACATAAAGCCTAAATAG-3′ 5′-CTATTTAGGCTTTATGTCCCTGAAGTAGGAACC-3′ 5′-GGTTCCTACTTCAGGGGCATAAAGCCTAAATAG-3′ 5′ CTATTTAGGCTTTATGCCCCTGAAGTAGGAACC-3′ (b) PCR Amplifications 5′-TCAAAGCTTACACCAGTCTTGTAAACC-3′ 5′-CTTACAAGCAAGTACAGCAATCAACC-3′ 5′-CCCACCCTTAACAGCACATAGTACA-3′ 5′-TGACCACCATCCTCCGTGA-3′ 5′-TCCGACATCTGGTTCCTACTTCA-3′ 5′-CGTGTGGGCTATTTAGGCTTTATG-3′ 5′-TGGTTAATAGGGTGATAGACCTGTGA-3′ 5′-TTGAGGAGGTAAGCTACATA-3′ (c) Primer Combinations forward primer
51 105 147 201 254 308 347 401
CA) using 20-µL reactions comprising 1× BD Advantage 2 PCR buffer (BD Biosciences Clontech, Palo Alto, CA), 500 nM each primer, 200 µM each dNTP, 1× BD Advantage 2 polymerase mix (BD Biosciences Clontech), and 2 µL of 1000 × diluted p16519T as target. Amplification comprised 35 cycles of 15 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C after initial denaturation at 95 °C for 2 min. Equal volumes of the self-priming SOE1 PCR products were pooled and nonincorporated primers removed by digestion with exonuclease I using the Exo-SAP-IT (Amersham Biosciences, Uppsala, Sweden) enzyme mix following the manufacturer’s instructions. In a second round of PCR amplification (SOE2), the complementary 3′ overlaps were extended at 72 °C for 5 min to generate full-length control regions containing bases C, A, or G at ntp 16 519, which were subsequently amplified with the outer primers p1F and p1272 in the same reaction mix by lowering the annealing temperature. The PCR was performed in a Gene Amp PCR System 9700 (Applied Biosystems) in a total volume of 20 µL containing 1× BD Advantage 2 PCR buffer (BD Biosciences Clontech), 300 nM each primer, 200 µM each dNTP, 1× BD Advantage 2 polymerase mix (BD Biosciences Clontech), and 2 µL 1000 × diluted, enzymatically treated, pooled SOE1 products. The thermal cycler protocol comprised an initial denaturation step of 2 min at 95 °C, 5 cycles of 95 °C for 15 s and 72 °C for 90 s, 24 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 90 s. The final extension step was carried out at 72 °C for 60 min. Unpurified SOE2 PCR products were ligated into the pCR4TOPO vector and transformed into chemically competent One Shot TOP10 Escherichia coli using the TOPO TA Cloning Kit for Sequencing (Invitrogen Life Technologies, Carlsbad, CA) following the manufacturer’s recommendations. Transformed cells were incubated at 37 °C overnight on agar plates containing 50 µg/mL
p594F p594F p498F p498F p391F p391F p298F p298F
reverse primer p644R p698R p644R p698R p644R p698R p644R p698R
kanamycin. Small portions of individual colonies were picked and transferred to 2 mL of LB medium supplemented with 50 µg/mL kanamycin. Plasmid DNA (pDNA) was isolated from the overnight (37 °C) suspension cultures using the monta´ge Plasmid Miniprep96 kit (Millipore, Billerica, MA) following the included instructions, and sequenced as described in ref 45 to confirm the success of the site-directed mutagenesis and to verify the absence of additional base substitutions. Aliquots of the plasmids were linearized by digestion with EcoRI (Roche, Basel, Switzerland) and quantified with the PicoGreen dsDNA Quantitation Kit (Molecular Probes, Eugene, OR) using λ-DNA as standard according to the manufacturer’s instructions. Based on these quantification results, circular plasmids were brought to equal concentrations by addition of buffer (10 mM TrisHCl, 0.1 mM Na2EDTA, pH 8.0 at room temperature), and the copy numbers were derived from the molecular masses of the vector and the respective insert. Equality in pDNA concentration for p16519A, C, G, and T was verified by SYBR Green I real-time detection PCR using primers p594F and p644R in an ABI PRISM 7700 sequence detector (Applied Biosystems). Polymerase Chain Reaction. To generate 51-401-bp-long amplicons 100 000 copies of pDNA were amplified with the primer pairs outlined in Table 1. PCR was conducted in a Gene Amp PCR System 9700 (Applied Biosystms) using 50-µL reactions comprising 1× BD Advantage 2 SA PCR buffer (BD Biosciences Clontech), 500 nM each primer, 200 µM each dNTP, 1× BD Advantage 2 polymerase mix (BD Biosciences Clontech), and 2 µL of diluted pDNA. Amplification comprised an initial denaturation step at 95 °C for 2 min, followed by 35 cycles of 15 s at 95 °C, 30 s at 60 °C, and 90 s at 68 °C. (45) Brandstatter, A.; Niederstatter, H.; Parson, W. Int. J. Legal Med. 2004, 118, 47-54.
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ICEMS. An Ultimate fully integrated capillary HPLC system (LC-Packings, Amsterdam, The Netherlands) was used for all chromatographic experiments. A Famos microautosampler (LCPackings) equipped with a 1-µL loop was used for sample injection. The 60 × 0.2 mm i.d. monolithic capillary column was prepared according to the published protocol.34 PCR amplicons were injected onto the column without any prior sample preparation step. ESI-MS was performed on a QSTAR XL mass spectrometer (Applied Biosystems) equipped with a modified TurboIonSpray source. The modifications included the replacements of the Peek tubing transfer line and of the stainless steel sprayer capillary by fused-silica capillaries (transfer line 375-µm o.d., 20-µm i.d.; sprayer capillary 90-µm o.d., 20-µm i.d.; Polymicro Technologies, Phoenix, AZ). The exit of the capillary column was connected directly to the transfer line by means of a microtight union (Upchurch Scientific, Oak Harbor, WA). Mass calibration and optimization of instrumental parameters were performed in the negative ion mode by infusion of a 1.75 pmol/µL solution of a 50-mer (5′-CCA GCC CTT CGA GAG GTC AAG GCA AGC GGA TCA CAG GGT GGA AGA GAT TG-3′, Eurogentec) in 25 mM aqueous BDMAB containing 20% acetonitrile (v/v) at a flow rate of 2.0 µL/min. Cations present in the oligonucleotide solution were removed by on-line cation exchange using a 20 × 0.50 mm i.d. cation-exchange microcolumn packed with 38-75-µm Dowex 50 WX8 particles (Serva, Heidelberg, Germany).46 The spray voltage was typically in the range of 3.0-3.5 kV. Gas flows of 10-15 (ion source gas 1) and 40 units (ion source gas 2) were employed. The temperature of ion source gas 2 was adjusted to 200 °C. The accumulation time was set to 1 s, and 10 time bins were summed up. Reconstructed ion chromatograms and mass spectra were recorded on a personal computer operating with the Analyst QS software (service pack 8, Applied Biosystems). Deconvolution of raw mass spectra was performed with Bayesian Protein Reconstruct (BioAnalyst 1.1.1, Applied Biosystems). All isotopic distributions were calculated with Isotopica.47 RESULTS AND DISCUSSION Fundamentals of Sequence Variation Detection by ICEMS. Like other mass spectrometric assays, ICEMS represents an elegant genotyping method due to the use of the molecular mass as a reliable indicator for the presence of sequence variations. While commonly used tracers such as retention or migration times can be influenced by experimental conditions, the molecular mass is an intrinsic property that is independent from the physical environment. Furthermore, as the allelic state derived from one single strand is confirmed by the result obtained from the complementary single strand, the reliability of the mass spectrometric genotyping assay is increased. The raw mass spectrum obtained from ESI-MS of a nucleic acid typically consists of a series of peaks, each of which represents a multiply charged ion of the intact nucleic acid having a specific number of protons removed from the phosphodiester groups. The signals of the multiply charged series can be readily deconvoluted to yield the corresponding molecular mass.48 To assign the allelic state(s), the measured molecular masses are (46) Huber, C. G.; Buchmeiser, M. R. Anal. Chem. 1998, 70, 5288-95. (47) Fernandez-de-Cossio, J.; Gonzalez, L. J.; Satomi, Y.; Betancourt, L.; Ramos, Y.; Huerta, V.; Amaro, A.; Besada, V.; Padron, G.; Minamino, N.; Takao, T. Nucleic Acids Res. 2004, 32, W674-8.
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Figure 1. Influence of mass accuracy on the detectability of sequence variations in homozygous samples (105-bp-long amplicon). Only one sequence variant is present within the sample.
compared to the theoretical molecular masses derived from the forward and reverse sequences of the known alleles. In this context, the mass spectrometric performance of the applied mass analyzer in regard to mass accuracy and resolution restricts the maximum allowable molecular masses and therefore lengths of nucleic acids for the unequivocal detection of base exchanges.41 Influence of Mass Accuracy and Resolution on the Detection of Sequence Variations in Homozygous Samples. A homozygous sample carries two identical alleles at the corresponding gene loci on homologous chromosomes. Therefore, all single PCR products are identical. In such a case, the mass accuracy plays the decisive role for the identification of the present allelic state. The resolution has only an auxiliary function. If the resolution is high enough to resolve the isotopic patterns, the molecular mass of a nucleic acid is determined by measuring its monoisotopic masses directly or via fitting a calculated to an experimentally determined isotopic distribution.49 The monoisotopic mass is defined as the mass of the isotopic peak whose elemental composition is assembled of the most abundant isotopes of the contained elements. At lower resolution, an envelope is detected, which encompasses the region of the isotopic peaks. In such a case, the m/z of the monoisotopic peak becomes undetectable and the average mass, which represents the average of the isotopic masses weighted by the individual isotopic abundances, is used for mass characterization. The experimentally determined mass is in such a case the top-of-centroid (TOC) mass, which experiences a negatively biased shift with respect to the average mass.50,51 Such a shift results from the presence of an asymmetric distribution of isotopomers, with peaks that contain a higher percentage of heavy isotopes forming a tail that extends toward high masses. The addition of 0.45 amu to measured masses was proposed to compensate the systematic error50,51 and was applied throughout this study to debug measured molecular mass values. The mass accuracy is affected by the accuracy and reproducibility of the molecular mass determination. A confidence interval around the true molecular mass of the investigated oligonucleotide represents the best illustration for this value (Figure 1). The (48) Mann, M.; Meng, C. K.; Fenn, J. B. Anal. Chem. 1989, 61, 1702-8. (49) Frahm, J. L.; Mason, C. J.; Muddiman, D. C. Int. J. Mass Spectrom. 2004, 234, 79-87. (50) Zubarev, R. A.; Demirev, P. A.; Haakansson, P.; Sundquist, B. U. R. Anal. Chem. 1995, 67, 3793-8. (51) Null, A. P.; Muddiman, D. C. Rapid Commun. Mass Spectrom. 2003, 17, 1714-22.
absolute size of the confidence interval depends on the magnitude of the molecular mass and the properties of the applied mass analyzer. To illustrate this point: if the TOC mass of one single strand of a 105-bp-long amplicon is 32 070.72 amu, a maximum mass deviation in the range of (200 parts per million (ppm), which is characteristic for quadrupole ion trap mass spectrometers,37,38,41 will be converted into a confidence interval of (6.41 amu. For the discrimination of the target sequence from a putative sequence variant, the confidence intervals around the molecular masses of these two species must not overlap. Unfortunately, the confidence intervals around the masses of the 105-mer and of a sequence, which was generated via the substitution of one A to a T, show an overlap (Figure 1). Consequently, the 105-mer cannot be unequivocally distinguished from the modified sequence via molecular mass measurements on a quadrupole ion trap mass spectrometer. Recently, it was experimentally demonstrated that based on the mass accuracy and resolution of quadrupole ion trap mass spectrometers the length of an oligonucleotide must not be longer than 70 bp in order to unequivocally detect all possible single base exchanges,41 which definitively restricts the usability of such an instrument for genotyping purposes. Extension of the size range, to which MS-based genotyping is applicable, is obtainable by implementation of a mass analyzer offering higher performance. Two different mass analyzers are known for their superior mass accuracy and resolution, namely, TOF and Fourier transform ion cyclotron resonance (FTICR) mass analyzers. Both instrument types were successfully applied for the characterization of nucleic acids.32,33,52-60 For both instruments, reported mass accuracies were in the range between a few ppm and 100 ppm. Hence, both instruments should be equally well suited for the differentiation of nucleic acid variants obtained from the PCR amplification of homozygous samples. Evaluation of the Performance of ESI-TOF-MS in Genotyping Homozygous Samples. A “SNP toolbox” was constructed by cloning and PCR-mediated site-directed in vitro mutagenesis at ntp 16519 of a sequence-verified fragment of the human mitochondrial genome (ntps 15 900-599). Exhaustive confirmatory sequencing demonstrated that there was no other base change in any of the used clones. Using this set of clones as PCR templates, different-sized amplicons were generated with the same base mutated to all other bases. These constructs were used to demonstrate the principle applicability of ICEMS in combination with a TOF instrument for the characterization of PCR products as well as to determine the upper limit of amplicon length for the unequivocal genotyping of SNPs in homozygous samples. (52) Hofstadler, S. A.; Sannes-Lowery, K. A.; Hannis, J. C. Mass Spectrom. Rev. 2005, 24, 265-85. (53) Little, D. P.; Thannhauser, T. W.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1995, 95, 2318-22. (54) Muddiman, D. C.; Wunschel, D. S.; Liu, C.; Pasa-Tolic, L.; Fox, K. F.; Fox, A.; Anderson, G. A.; Smith, R. D. Anal. Chem. 1996, 68, 3705-12. (55) Null, A. P.; Benson, L. M.; Muddiman, D. C. Rapid Commun. Mass Spectrom. 2003, 17, 2699-706. (56) Laken, S. J.; Jackson, P. E.; Kinzler, K. W.; Vogelstein, B.; Strickland, P. T.; Groopman, J. D.; Friesen, M. D. Nat. Biotechnol. 1998, 16, 1352-6. (57) Ni, J.; Chan, K. Rapid Commun. Mass Spectrom. 2001, 15, 1600-8. (58) Koomen, J. M.; Russell, W. K.; Tichy, S. E.; Russell, D. H. J. Mass Spectrom. 2002, 37, 357-71. (59) Flora, J. W.; Null, A. P.; Muddiman, D. C. Anal. Bioanal. Chem. 2002, 373, 538-46. (60) Muhammad, W. T.; Fox, K. F.; Fox, A.; Cotham, W.; Walla, M. Rapid Commun. Mass Spectrom. 2002, 16, 2278-85.
Figure 2. Genotyping of the locus 16519 by analyzing 51-bp-long amplicons. Column, monolithic PS-DVB, 60 × 0.20 mm i.d.; mobile phase, (A) 25 mM BDMAB, pH 8.40, (B) 25 mM BDMAB, pH 8.40, 40% acetonitrile; linear gradient, 5-50% B in 10 min; flow rate, 2.0 µL/min; temperature, 68 °C; scan, 800-1800 amu; sample, 1.0 µL of PCR solution containing 51-bp-long amplicons of the locus 16519.
In Figure 2, the results of the analysis of a 51-bp-long PCR product of the allele C are shown. The amplicons were chromatographically separated from the remaining components of the PCR solution, thermally denatured into the corresponding single strands, and detected by ESI-TOF-MS. During the first 4 min of the run, the sprayer was shifted from its optimal position to a more peripheral position to reduce the degree of contamination of the ion-transfer optics with components of the PCR buffer. The two single strands coeluted after 6.6 min (Figure 2a). The raw mass spectrum (Figure 2b) was extracted from the peak in the reconstructed ion chromatogram (RIC). A simple visual inspection of the raw mass spectrum revealed the presence of three to four different species. The corresponding molecular masses were obtained by applying a deconvolution algorithm. The mass information was used to identify, in fact, four different oligonucleotides. The measured molecular masses correlated well to the theoretical masses of the two single strands and of species arising from the nontemplate addition of deoxyadenosine as well as of deoxyguanosine to the forward single strand (inset in Figure 2b, Table 2). In similar ways, amplicons of the three other alleles were analyzed. In all cases, the relative mass deviations were smaller than (64.1 ppm, which enabled the unequivocal calling of the present allelic states (Table 2). In a further set of experiments, the amplicon size was extended starting from 51 bp up in steps of ∼50 bp (Table 1c) to determine the upper length limit for the analyzability of PCR products and for the detectability of sequence variations, respectively. Along with size, the molecular masses and the number of observed charge states increased. Interestingly, the relative mass deviation Analytical Chemistry, Vol. 77, No. 15, August 1, 2005
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Table 2. Genotyping of All Four Possible Alleles of 16 519 by Comparison of Theoretical and Measured Molecular Massesa measd mass (amu) 15 547.67 15 840.94 15 861.35 15 877.83 15 587.71 15 800.31 15 900.70 15 571.72 15 815.82 15 885.06 15 900.68 15 562.87 15 824.11 15 875.76 15 892.30 a
corrected mass (amu) assignment 15 548.12 15 841.39 15 861.80 15 878.28 15 588.16 15 800.76 15 901.15 15 572.17 15 816.27 15 885.51 15 901.13 15 563.32 15 824.56 15 876.21 15 892.75
Cfor Crev Cfor + dA Cfor + dG Gfor Grev Gfor + dA Afor Arev Afor + dA Afor + dG Tfor Trev Tfor + dA Tfor + dG
theor mass (amu)
relative deviation (ppm)
15 548.06 15 841.21 15 861.27 15 877.27 15 588.08 15 801.19 15 901.29 15 572.08 15 816.20 15 885.29 15 901.29 15 563.07 15 825.21 15 876.29 15 892.28
3.8 11.4 33.5 64.1 5.1 -27.1 -9.0 5.2 4.7 13.9 -10.1 16.0 -41.6 -4.5 30.0
allele ID 16 519 C
16 519 G 16 519 A
16 519 T
Experimental conditions as described in Figure 2.
was unaffected from oligonucleotide length and was in all cases below (55.0 ppm. The single strands of the 401-mer were found to be the largest DNA fragments, of which interpretable mass spectra were obtained. As shown in Figure 3, due to the complexity and the low signal-to-noise ratios, the raw mass spectra of such large nucleic acids with molecular masses in the range of 122 000-126 000 amu were manually uninterpretable. However, the implemented software transferred these raw mass spectra into deconvoluted mass spectra, in which four different species were unequivocally identified (insets in Figure 3b,c and Table 3). For the assignment of the allelic state, the obtained molecular masses were compared to the theoretical masses calculated from the sequences of the four possible alleles (Table 3). The overall smallest mass differences were obtained for allele A, which in point of fact was the correct allele. A systematic error within the molecular mass measurements was indicated by collectively positive mass deviations in the range between 16.1 and 44.5 ppm. No single sequence variation would cause a simultaneous increase of the molecular masses of both single strands. Due to the paring of one lighter and one heavier nucleotide (A (313.207 amu) and T (304.194 amu) as well of as G (329.207) and C (289.183)) within double-stranded DNA molecules, any single base exchange within the forward strand sequence that causes a shift of the molecular mass in one direction induces a shift of the molecular mass of the complementary strand in the opposite direction. Hence, the correction of the measured molecular masses was permissible and was accomplished by detracting the average mass deviation of ∼30 ppm from each measured value (Table 3). The debugged molecular masses reinforced the initially obtained genotyping result, which clearly proves the importance of measuring the molecular masses of both single strands to increase the reliability of allele calling. The simultaneous determination of the molecular masses of complementary single strands represents an elegant method for the identification and correction of systematic errors, which reduces the need for any other internal calibration procedure. Influence of Mass Accuracy and Resolution on the Detection of Sequence Variations in Heterozygous Samples. In all above-described experiments the detectability of sequence varia5004 Analytical Chemistry, Vol. 77, No. 15, August 1, 2005
Figure 3. Analysis of a 401-bp-long PCR product. Mobile phase, (A) 25 mM BDMAA, pH 8.30,w r, (B) 25 mM BDMAA, pH 8.30, 40% acetonitrile; linear gradient, 5-75% B in 12.5 min; scan, 800-1150 amu; sample, 1.0 µL of PCR solutions containing 401-bp-long amplicons of the locus 16 519. Other experimental conditions as described in Figure 2.
tions was evaluated via the analysis of PCR amplicons obtained from homozygous samples. The human genome is diploid, and the simultaneous presence of two sequence variants needs to be considered. For unequivocal genotyping, the mass spectrometric performance must be high enough to resolve the two species or at least to unequivocally distinguish the heterozygous state from the two homozygous states. In general, a resolution power of 20 000-25 000 (fwhm) is achievable on TOF instruments. Nevertheless, PCR products are usually represented by rather broad and asymmetric peaks in the deconvoluted mass spectrum (Figure 4a). Every single peak emblematizes an envelope covering the entire unresolved isotopic profile. The principle broadness of such an envelope is governed by the number of naturally occurring isotopes and their individual abundances. Depending on the mass spectrometric sensitivity, which influences the noise level in the deconvoluted mass spectrum, a smaller or larger section of a single envelope is detectable. If two species are simultaneously present with a molecular mass difference smaller than approximately half of the observed peak widths, their envelopes will overlap (Figure 4a).
Figure 4. Influence of mass accuracy and resolution on the detectability of the correct allelic state of heterozygous samples (254bp-long amplicons). Two sequence variants are present within the sample. Table 3. Genotyping of a Homozygous Sample by Analyzing a 401-bp-Long Amplicona measured massb (amu)
assignment
theor mass (amu)
relative deviationb (ppm)
122 412 (122 408) 125 242 (125 238) 122 722 (122 718) 125 558 (125 554)
Afor Arev Afor + dA Arev + dA
122 407 125 239 122 720 125 552
42.4 (12.6) 22.3 (-7.4) 16.1 (-13.6) 44.5 (14.8)
122 412 (122 408) 125 242 (125 238) 122 722 (122 718) 125 558 (125 554)
Tfor Trev Tfor + dA Trev + dA
122 398 125 248 122 711 125 561
116.0 (86.3) -49.7 (-79.4) 89.6 (59.9) -27.3 (-57.0)
122 412 (122 408) 125 242 (125 238) 122 722 (122 718) 125 558 (125 554)
Cfor Crev Cfor + dA Crev + dA
122 383 238.7 (209.0) 125 264 -177.4 (-207.1) 122 696 211.9 (182.2) 125 577 -154.7 (-184.4)
122 412 (122 408) 125 242 (125 238) 122 722 (122 718) 125 558 (125 554)
Gfor Grev Gfor + dA Grev + dA
122 423 -88.3 (-118.0) 125 224 142.2 (112.5) 122 736 -114.3 (-144.0) 125 537 164.1 (134.4)
allele ID 16 519 A
a Experimental conditions as described in Figure 3. b Debugged molecular masses and relative deviations are listed in parentheses, respectively.
As long as two apexes will be observed, the unequivocal identification of both alleles present within a heterozygous sample is possible. Above a certain molecular mass value, the two peaks will merge into one single peak (Figure 4a) with a TOC mass representing nearly the median of the two allele-specific masses. The signal shape cannot be used anymore to distinguish hetero-
and homozygous samples. Under these circumstances, molecular mass measurements, which enable the accurate and reproducible differentiation of the three characteristic TOC masses, are necessary for the identification of the present allelic state(s) (Figure 4a). FTICR instruments offer a comparably higher resolution power than TOF instruments. At 7-9.4 T a resolution of more than 150 000 (fwhm) was reported for DNA molecules52,61 and of more than 1 000 000 (fwhm) for other molecules.62 As shown in Figure 4b, such an ultimate resolution would enable the complete separation of the isotopic peaks of a 254-mer with a molecular mass of ∼77 670 amu. The principle shape of the isotopic pattern is governed by the number of naturally occurring isotopes and their individual abundances. If two species are present with a molecular mass difference smaller than approximately half of the widths of their isotopic patterns, the isotopic distributions will overlap (Figure 4b). For the individual identification and differentiation of the two overlapping isotopic distributions, all isotopic peaks would have to be resolved from each other regardless of their affiliation. Unfortunately, as shown in Figure 4b, isotopic peaks corresponding to the two different alleles are practically congruent with each other (isobar). At a molecular mass of 77 670 amu, mass differences of around (0.02 amu were calculated making a resolution unexecutable even with the ultimate resolving power offered by FTICR instruments. Hence, the two isotopic distributions merge into one broad pattern (Figure 4b). The most abundant isotopic peak has a molecular mass representing nearly the median of the masses of the most abundant isotopic peaks of the two individual alleles. In such cases, molecular mass measurements, which enable the accurate and reproducible differentiation of the mass of the most abundant isotopic peak of the heterozygous case from the masses of the most abundant isotopic peaks of the two homozygous states, are necessary to differentiate heterozygous and homozygous samples (Figure 4b). Hence, for large amplicon lengths, genotyping of heterozygous samples is based on precise and accurate molecular mass measurements. The resolving power has only an auxiliary function, which can help to improve the accuracy of molecular mass measurements but does not enable the direct differentiation of two simultaneously present nucleic acid species. Based on these theoretical considerations, we believe that TOF and FTICR instruments should be equally well suited for the genotyping of heterozygous samples. Evaluation of the Performance of ESI-TOF-MS in Genotyping Heterozygous Samples. The SNP toolbox was applied for the evaluation of the usability of the TOF analyzer for the genotyping of heterozygous samples. Heterozygous states were prepared by mixing equimolar amounts of clones of different alleles. In a first set of experiments, clones of all four alleles were combined to one sample and 51-bp-long PCR products were prepared. The obtained reaction products were characterized in three consecutive analyses. In each experiment, the amplicons from 1.0 µL of crude PCR solution were chromatographically purified and thermally denatured. The eluting DNA fragments were detected by ESI-TOF-MS. The extracted raw mass spectra (61) Benson, L. M.; Null, A. P.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2003, 14, 601-4. (62) Cole, R. B. Electrospray Mass Spectrometry: Fundamentals, Instrumentation & Applications; John Wiley & Sons: New York, 1997.
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Figure 5. Repeated genotyping of a sample containing all four possible alleles of the locus 16 519. Mobile phase, (A) 25 mM BDMAB, pH 8.40, (B) 25 mM BDMAB, pH 8.40, 40% acetonitrile; linear gradient, 5-50% B in 10 min; scan, 800-1500 amu; samples, 1.0 µL of PCR solutions containing 51-bp-long amplicons of the locus 16519. Other experimental conditions as described in Figure 2. Table 4. Allele Calling in Heterozygous Samples via the Measurement of the Molecular Masses of 254-bp-Long Ampliconsa measd mass (amu) 77 648 77 689 79 134 79 175 77 648 77 676 79 147 79 180 77 677 77 687 79 136 79 147 77 669 79 153 a
assignment
theor mass (amu)
relative deviation (ppm)
Cfor Gfor Grev Crev Cfor Afor Arev Crev Afor Gfor Grev Arev Tfor Afor Arev Trev
77 650 77 690 79 134 79 174 77 650 77 674 79 149 79 174 77 674 77 690 79 134 79 149 77 665 77 674 79 149 79 158
-25.8 -12.9 0.0 12.6 -25.8 25.7 -25.3 75.8 38.6 -38.6 25.3 -25.3 51.6 -64.3 50.2 -63.5
allele ID 16 519 C/G
16 519 A/C
16 519 A/G
16 519 A/T
Experimental conditions as described in Figure 7.
were deconvoluted. The resulting deconvoluted mass spectra are depicted in Figure 5. Capital letters were used as abbreviations representing the nucleotide present at the polymorphic site of the forward strand. The mixture consisted of four different alleles. Accordingly, eight different single-stranded species were detected. Although the theoretical masses of two on the molecular mass scale adjacent sequences differed by only 9.01 (T and A), 15.01 amu (C and T), and 16.00 amu (A and G) at an overall molecular mass of ∼15 500 amu, all species were effortlessly separated from each other. Single molecular masses were measured with high accuracy and precision. In all cases, the mean molecular masses deviated not more than (18.4 ppm from the theoretical values. Interestingly, the nucleotide at the polymorphic site had an impact on the relative signal intensity of the corresponding oligonucleotide. Somehow, the exchange of a single nucleotide altered the ionization efficiencies of otherwise identical oligonucleotides. The signal height decreased in the order A g T . G g C (Figure 5), which needs to be considered in the context of semiquantitative genotyping.42 To determine the upper size limit of PCR products for the detection of all kinds of single base exchanges in heterozygous samples, representative allelic mixtures were amplified in 147- and 254-bp-long amplicons and analyzed by ICEMS. The deconvoluted 5006
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mass spectra obtained from the analyses of samples containing 147-bp-long amplicons are depicted in Figure 6. The components of the three allelic mixtures, G/C, A/G, and A/C, were resolved from each other. Based on the molecular mass information, the corresponding alleles were called. The mass deviations were in all cases smaller than (29.8 ppm. For the mixture of the alleles A and T, four broad instead of eight narrow peaks were observed (Figure 6d). None of these peaks was attributable to one certain allele. For every single strand, the measured molecular mass represented nearly the average of the theoretical molecular masses of the two considered alleles. An overlay of the deconvoluted mass spectrum of the heterozygous sample with the deconvoluted mass spectra of homozygous samples containing only allele A or allele T, respectively, clearly supported the hypothesis that the observed peaks did not represent a single allele (Figure 6e, f). In fact, the peaks reflected the unresolved mass signals of two simultaneously present alleles indicating a heterozygous sample. Hence, also for the A/T polymorphism, the heterozygous state was unequivocally distinguishable from the homozygous states. Accordingly, at the 150-mer level, mass spectrometric genotyping results are unambiguous. In a further set of experiments, the mass spectrometric detectability of individual alleles in heterozygous samples was evaluated by analyzing 254-bp-long amplicons (Table 4, Figure 7). The allele-specific signals of all allele combinations except A/G and A/T were separated. In the deconvoluted mass spectrum obtained from the analysis of the sample containing the alleles A and G, the presence of a second allele was indicated by shoulders on the principle peaks (Figure 7a, b). The mass information extracted from the peaks and their shoulders enabled the unequivocal identification of both alleles (Table 4). In the case of the heterozygous sample, which contained allele A and T simultaneously, two instead of the expected four signals were observed. For each single strand, the peaks, which correspond to the two alleles, merged into one broad peak (Figure 7d, e) with a molecular mass representing nearly the median of the two individual allelic masses (Table 4). Due to the length of the amplicons, the obtained relative mass deviations came close to the maximum deviations observed for the analysis of homozygous samples (Table 4). Hence, there was some chance of miscalling. However, since none of the measured masses fitted well together with the theoretical masses of one of the two homozygous states, the probability of misinterpretation was small. Therefore, also for
Figure 6. Genotyping of samples containing different combinations of the four possible alleles of the locus 16 519 amplified in 147-bp-long amplicons. Mobile phase, (A) 25 mM BDMAA, pH 8.30, (B) 25 mM BDMAA, pH 8.30, 40% acetonitrile; linear gradient, 5-50% B in 10 min; scan, 800-1500 amu; samples, 1.0 µL of PCR solutions containing 147-bp-long amplicons of the locus 16519. Other experimental conditions as described in Figure 2.
the A/T polymorphism, the differentiability of homo- and heterozygous samples was possible at the 250-mer level. For the characterization of an A/T polymorphism, the use of longer amplicons is not advisable. Since the mass measurement error would become larger than the mass deviation of the unresolved A/T from the homozygous states, the discrimination between A, T, and A/T becomes inexecutable. Hence, the upper size limit for the unequivocal genotyping of an A/T polymorphism in samples from diploid genomes by ESI-TOF-MS is at ∼250 bp. Nevertheless, all other single base substitutions can be successfully genotyped by analyzing even larger nucleic acids. CONCLUSIONS Limitations for ESI-MS-based mass spectrometric genotyping in regard of the maximum allowable amplicon length for the unequivocal detection of base exchanges arise from the performance of the applied mass analyzer. Two different instrument
types are known for their superior mass spectrometric performance, namely, TOF and FTICR mass analyzers. Both enable highly accurate molecular mass measurements with errors in the range of a few to 100 ppm, and both offer resolving powers far beyond 10 000, enabling the resolution of the isotopic patterns of rather large oligonucleotides. Therefore, both instruments are highly suited for the mass spectrometric characterization of nucleic acids. It is well known that FTICR instruments offer a comparably higher resolving power than TOF instruments. However, our theoretical considerations clearly proved that even the high resolving power of FTICR instruments does not enable a further differentiation of two simultaneously present alleles within heterozygous samples in cases where the TOF analyzer would fail. Depending on the relative mass difference, the isotopic distribution corresponding to one allele can overlap with the pattern of the other allele. Above a characteristic amplicon length, the two Analytical Chemistry, Vol. 77, No. 15, August 1, 2005
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Figure 7. Genotyping of samples containing the alleles (a, b) A/G and (c, d) A/T, respectively, amplified in 254-bp-long amplicons. Mobile phase, (A) 25 mM BDMAA, pH 8.30, (B) 25 mM BDMAA, pH 8.30, 40% acetonitrile; linear gradient, 5-50% B in 10 min; scan, 800-1500 amu; samples, 1.0 µL of PCR solutions containing 254-bp-long amplicons of the locus 16 519. Other experimental conditions as described in Figure 2.
independent distributions merge into one broad pattern. Accordingly, the signal shape cannot be used to distinguish hetero- and homozygous samples. In such cases, molecular mass measurements, which enable the accurate and reproducible differentiation of the molecular mass obtained from the heterozygous case from the masses of the two homozygous states, are necessary to differentiate heterozygous and homozygous samples. Hence, genotyping of large amplicons is based on precise and accurate molecular mass measurements. The resolving power has only an auxiliary function, which can help to improve the accuracy of molecular mass measurements but does not enable the direct differentiation of two varying nucleic acid sequences. Accordingly, TOF and FTICR instruments should be equally well suited as genotyping tools. However, since TOF instruments are more convenient for routine work due to their comparably lower purchase and operating costs and their easier handling, we propagate the use of this type of instrument for nucleic acids analysis. The high usability of TOF instruments for mass spectrometric genotyping was proven experimentally. Based on the offered mass
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spectrometric performance, the detection of all kind of single base exchanges was feasible in PCR amplicons from diploid genomes with lengths up to ∼250 bp. Considering haploid markers, the high mass accuracy of the TOF mass analyzer enabled the genotyping of single base substitutions in up to ∼400-bp-long PCR products. As far as we know, a comparable performance in nucleic acid analysis has not been reached by any other mass spectrometric method before. Even for high-performance ESI-FTICR-MS, it was proclaimed that it is not applicable to the genotyping of amplicons with lengths beyond 200 bp.63 Definitively, LC-ESI-TOFMS represents one of the most powerful mass spectrometric genotyping assays available today, which has great potential to become a very popular method for genome research soon. Received for review March 7, 2005. Accepted May 11, 2005. AC050399F (63) Van Ert, M. N.; Hofstadler, S. A.; Jiang, Y.; Busch, J. D.; Wagner, D. M.; Drader, J. J.; Ecker, D. J.; Hannis, J. C.; Huynh, L. Y.; Schupp, J. M.; Simonson, T. S.; Keim, P. Biotechniques 2004, 37, 642-8.
