Published: September 20th, 2016
An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.
An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 ± 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.
Mass spectrometry (MS) is an accepted technology for rapid and reliable identification of nucleic acids, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) being used for ionization1,2. The MALDI technique is typically combined with a time-of-flight (TOF) analyzer; however, the application of MALDI-TOF MS is limited to short oligonucleotides (~25 nucleotides (nt)) owing to the subsequent fragmentation, adduct formation and low ionization efficiency1,2. In contrast, ESI is applicable to longer oligonucleotides (>100 nt), but many charge states of multiple charged ions ([M−nH]n−) are produced simultaneously from biomacromolecules and, therefore, the mass of the analyte can exceed the upper limit of the m/z range of the spectrometer. This requires the interpretation of the convoluted spectra, i.e., transformation of a charge state series into the corresponding mass via deconvolution.
In the case of mass measurement of a small molecule, the peak of the monoisotopic mass, i.e., the mass of the molecule containing only the most common isotope of each element is the most abundant and observed naturally3. As the molecular weight increases, the isotopic distribution shifts to higher m/z and the monoisotopic peak becomes obscured by baseline noise3-5. Once the monoisotopic peak is no longer detectable for masses greater than 10 kDa3, the average molecular mass is used for measurement rather than the monoisotopic mass5. In such cases, the isotopic distribution in which each of the individual peaks is separated by 1 Da can only be observed when a high resolution mass analyzer such as a TOF, a Fourier transform modified Kingdon trap analyzer6, or a Fourier transform ion cyclotron resonance analyzer is used for analysis. However, the most abundant peak is sometimes not consistent with the average molecular mass5. These problems can lead to difficulty for accurately determining analytes.
Given the variation in the natural abundance of stable isotopes and the difficulties in the determining the average molecular mass, measurement of the monoisotopic mass is ideal for mass characterization of biomolecules3,4. In practice, whether a monoisotopic peak can be observed or not, the monoisotopic mass can be estimated by comparing the pattern of the observed isotopic distribution to the theoretical one calculated from a model analyte4,7-10. The fitting algorithm8 is now incorporated into proprietary software.
In the context of ESI-MS, dissociation of a DNA duplex, purification and desalting are required for direct measurement to avoid the failure of ionization due to ion suppression and adduct formation2,10-14. These procedures are troublesome, however, fully automated analytical systems have been developed commercially involving polymerase chain reaction (PCR), sample preparation and ESI-MS for the detection of pathogens15-20. Another approach is introducing liquid chromatography (LC) for separation. LC provides an on-line separation of the analytes from coexisting substances and does not require a laborious sample preparation prior to ionization21,22. However, separation of nucleic acids using a MS-compatible mobile phase is more difficult than for most other compounds such as drugs, peptides and proteins owing to the polyanionic nature of the nucleotides. The most successful examples of LC/ESI-MS involve the use of ion-pair reverse-phase LC. A mobile phase of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)-triethylamine (TEA)-methanol was initially proposed by Apffel et al. for separating and detecting short oligonucleotides23. Application of LC/ESI-MS genotyping for differentiation of pathogen species, single-nucleotide polymorphisms and short tandem repeats has been reported using a capillary polymer-monolith column that can separate longer oligonucleotides1,21,24-33.
In Japan and the United States, serious food poisoning has occurred due to misidentification and inappropriate preparation of pufferfish, and this is despite the fact that the distribution and preparation of pufferfish is strictly controlled by food safety legislation34,35. Moreover, an intentional homicide using pufferfish extract did occur in Japan36. Therefore, differentiation of pufferfish species is required from both public health and forensic investigative standpoints. Additionally, because Takifugu rubripes is far more expensive than other kinds of pufferfish, a differentiation capability is also needed in the context of food fraud investigations.
Here, a detailed method for determining the monoisotopic mass of a PCR product by LC/ESI-MS using a reverse-phase silica monolith column and a high resolution mass spectrometer is described. Specifically, the approach has been developed to permit differentiation of toxic pufferfish species based on the use of the PCR restriction fragment length polymorphism (RFLP) method37, which is the first example for species differentiation of animals using MS.
1. DNA Extraction
Note: Use a separate room for DNA extraction and post-PCR examinations such as gel electrophoresis and LC/ESI-MS. Add ethanol to wash buffer 1 (containing guanidine hydrochloride) and wash buffer 2 (not containing guanidine hydrochloride) according to the DNA extraction kit protocol. Fish samples were obtained from fish wholesalers and retail markets in Japan.
3. Enzymatic Digestion
4. LC/ESI-MS Analysis
5. Deconvolution and Interpretation
Three commercially-available columns were evaluated for the separation of long oligonucleotides at flow rates of 100-400 µl/min. A wide-pore octadecyl carbon chain (C18)-bonded particulate silica column (a), a commercially-available poly(styrene-divinylbenzene) (PS-DVB) monolith column (b) and a C18-bonded silica monolith column (c) were compared (Figure 1). Three pairs of the DNA duplexes (26, 37 and 53 bp) were separated using all the columns, and the C18-bonded silica monolith column was used in subsequent studies.
Representative results for the analysis of a DNA sample extracted from the muscle of T. rubripes is shown in Figure 2. Isotopically resolved peaks, as shown in Figure 2a, are required for successfully calculating the monoisotopic mass. The theoretical and measured monoisotopic mass of T. rubripes are shown in Figure 2b. Because the digestion with the endonucleases is performed just after PCR amplification without purification, the 3ʹ-sticky end generated by the Msp I endonuclease is filled up with the remaining DNA polymerase. According to the analysis of amplicons derived from the synthetic DNA templates, mass accuracy ranged from -2.48 to 2.40 ppm (0.62 ± 0.74 ppm on average, n = 280). Thus, a mass tolerance of 3 ppm was used for differentiating species (Table 4). Using the table, all of the pufferfish samples obtained from markets and stores were properly differentiated as specific species37.
According to the periodic analysis of the sample obtained from the synthetic DNA template of T. chrysops, all the monoisotopic masses of the analytes were successfully determined within ± 3 ppm for at least 180 hr without any mass calibration (Figure 3). This means that mass calibration would be required on a weekly basis.
Figure 1: Total ion current chromatograms of the PCR-RFLP product derived from the synthesized DNA template of T. pardalis using a C18-bonded particulate silica column (a, 3×250 mm, particle size 3 µm), a poly(styrene-divinylbenzene) (PS-DVB) monolith column (b, 1.0×250 mm) and a C18-bonded monolith silica column (c, present method). Conditions for (a): flow rate 0.2 ml/min; ratio of methanol, 0-2 min 5%, 2-5 min 5%-25%, 5-20 min 25%-40%, 20-35 min 40%-98%, 35-50 min 98%, 50-51 min 98%-5%, 51-65 min 5%. Conditions for (b): flow rate 0.1 ml/min; ratio of methanol, 0-2 min 5%, 2-5 min 5%-25%, 5-35 min 25%-40%, 35-40 min 40%-98%, 40-55 min 98%, 55-56 min 98%-5%, 56-70 min 5%. The temperature for both columns was 20 °C. Please click here to view a larger version of this figure.
Figure 2: Representative results for analyzing raw muscle of T. rubripes. (a) Typical total ion current chromatograms and mass spectrum. (b) Theoretical and measured monoisotopic masses of the PCR-RFLP products derived from the synthesized DNA template of T. rubripes. Please click here to view a larger version of this figure.
Figure 3: Stability test. Digested amplicons derived from the synthetic DNA of T. chrysops were analyzed every 10 hr. Mass calibration was not performed during the test. Please click here to view a larger version of this figure.
Table 1: PCR primers.
|Detergent-free 5x Buffer
|dNTP mix (10 mM each)
|Primer mix (10 μM each of lago86F, taki114F and fugu86-114R in TE buffer pH 8.0)
|0.4 μM each
|DNA polymerase (2.5 unit/μl)
|Template DNA in TE buffer pH 8.0 (5.0–10 ng/μl for extracted sample, 0.2 μM for synthetic DNA as positive control)
|0.5-1.0 ng/μl for extracted DNA and 20 nM for synthetic DNA
|Total: 25 μl
Table 2: Components of a 25 µl scale PCR.
|2 min at 95 °C
|30 s at 95 °C
|30 s at 56 °C
|30 s at 72 °C
|Repeat 2-4 (totally 30 cycles)
|7 min at 72 °C
|Hold at 12 °C until removal of the sample
Table 3: PCR program.
|Peak number at retention time of 4.0-5.5 min
|Monoisotopic mass of the third peak in the range (Da)
|Monoisotopic mass of the second peak in the range (Da)
Table 4: Differentiation of pufferfish from monoisotopic masses derived from LC/ESI-MS.
Table 5: Amplified DNA sequence. (a) The sequence is identical to that of the other pufferfish species as described in Table 4. Please click here to view a larger version of this table.
To extract DNA, a commercial kit for extracting DNA from blood and tissues is used in accordance with the protocol of the manufacturer with minor modification (amount of proteinase K and centrifugation of the lysis solution). However, any extraction kit can be used as long as cellular DNA can be extracted with appropriate recovery and purity for PCR. This method has been tested using muscle, fin, liver, ovary, and skin37. Fin is especially suitable because of the large surface area, which enables rapid lysis with proteinase K. Fresh, frozen, dried, boiled and baked pufferfish samples were tested successfully37.
The PCR primer set (Table 1) is designed for pufferfish and amplifies the mitochondrial 16S ribosomal RNA gene of pufferfish. The target DNA to amplify is 114-115 bp (genus Takifugu) and 86 bp (genus Lagocephalus) (Table 5). To facilitate method development, synthesized oligonucleotides having the target sequences were used for the reference instead of collecting authentic pufferfish specimens. The primer set can be replaced by another primer set in response to the purpose, however, final DNA length after enzymatic digestion should be less than 100 nt in terms of maintaining the quality of the mass spectrum required for successful deconvolution. Additionally, nitrogen pressure in the C-trap should be reduced when the target oligonucleotide is longer than about 75 nt and the quality of the mass spectrum is insufficient for successful deconvolution. Such limitations can be controlled through recent developments in instrument software, otherwise the valve for the C-trap inside the chassis must be tuned as described in the protocol section. As for sample preparation, use of detergent-free reagents is critical for the subsequent LC in terms of appropriate peak shape, sufficient peak intensity and stable retention time31.
A PS-DVB capillary monolith column21,24-33, a C18 reverse-phase particulate silica column23,40,41 and a hydrophobic interaction chromatography (HILIC) column42 have been used for the LC/ESI-MS analysis of oligonucleotides. Among them, the capillary monolith column is superior to the others in terms of separation capacity, however, the capillary monolith column used in past studies was made in-house and operated at a low flow rate (2 µl/min), which requires instrumentation dedicated to micro LC. To facilitate easy operation, commercially-available columns were evaluated for the separation of long oligonucleotides at higher flow rates (100-400 µl/min). Three pairs of DNA duplexes (26, 37 and 53 bp) were separated using the above-mentioned columns, however, the cycle times of the C18-bonded particulate silica column and the PS-DVB monolith column were 65 and 70 min, respectively, whereas that of the C18-bonded silica monolith column was 8 min (Figure 1). Taking rapid analysis into consideration, the C18-bonded silica monolith column was chosen for our purposes despite the limited separation capacity; however the remaining two columns may be employed when improved separation is required. Theoretically, in the case of a monolith column, there is no interstitial volume and the mobile phase would be forced to flow through the pores of the solid phase while maintaining a consistent path length, thus enabling an efficient separation27. Such processes would be manifested, particularly in the analysis of biomacromolecules such as an oligonucleotide, as the slow diffusion of the large molecules. One of the practical merits of a monolith column is that the back pressure is lower than that of a particulate silica column27. Despite the high flow rate (400 µl/min) and low column temperature (20 °C), maximum back pressure of the system was 12.5 MPa37. This is the first demonstration of the advantage of a C18-bonded silica monolith column for the rapid analysis of a long oligonucleotide. Owing to the high flow rate, a dedicated instrument for micro LC and precise alignment at the interface are not required. Instead, a heated ESI probe is required to dissociate the DNA duplex and assist ionization of DNA as described later.
Ion-pair chromatography is commonly used for the MS-compatible separation of oligonucleotides. However, an ion-pair reagent generally interferes with the ESI process and decreases sensitivity of ESI-MS. Therefore, HFIP is frequently used for the mobile phase to improve the sensitivity of the oligonucleotide. However, HFIP (boiling point 59 °C) vaporizes rapidly at the interface before methanol (boiling point 65 °C) and, therefore, this loss of solvent increases pH and promotes dissociation of the ion-pair reagent (i.e., TEA) from the oligonucleotide. Because the present method employs a heated ESI probe, which nebulizes the eluate with hot nitrogen gas at 350 °C, this effect may be over-emphasized. Instead of HFIP-TEA buffer, Erb and Oberacher recommended cyclohexyldimethylammonium acetate (CycHDMAA; pH 8.4) for genotyping analysis because of a reduction in adduct formation with trace metal ions33. The authors deduced that CycHDMAA itself suppressed the formation of the metal adduct. Despite the literature, significant adduct formation has not been observed in the present method. Additionally, the noteworthy benefit of the HFIP-TEA-methanol system is that the peak area obtained with the HFIP-TEA-methanol system was 17 times greater than that obtained from the CycHDMAA-acetonitrile system when analyzing an 86 bp amplicon of T. poecilonotus (data not shown). One disadvantage of the HFIP-TEA-methanol system, however, is the increased cost relative to the CycHDMAA-acetonitrile system.
Calculation of the monoisotopic mass requires the separation of isotopic peaks of multiply charged ions. Therefore, resolution is critical for the present analysis. Although requisite resolution power is dependent on the base pair length of the analyte, conventional TOF analyzers, which have a resolution power of several tens of thousands, may be limited for the analysis of short oligonucleotides.
The theoretical monoisotopic masses shown in Table 4 were calculated from the corresponding base compositions of the analytes. Alternatively, Muddiman et al. developed a software application to calculate the base composition from the accurate mass43. A similar program was integrated into the automated ESI-MS system16-18. The use of these algorithms may improve the robustness of the present method because a measured monoisotopic mass does not always correspond to a unique base composition owing to the mass tolerance of 3 ppm resulting from the inevitable measurement error. Unfortunately, we could not obtain these software products for the present study.
The present monoisotopic mass-based species determination may be suitable not only for the differentiation of pufferfish but also for the detection of other DNA polymorphisms because the present method is based on analyzing the base composition and does not involve a procedure specifically designed for pufferfish. As for the detection of DNA polymorphism, the dedicated ESI-MS system is fully automated and easy to operate, which may be suitable for diagnostic use such as detection of pathogens15,17,18,20,30. Conversely, the present method is feasible with common research instruments and apparatus and, therefore, suitable for research uses. ESI-MS has already been applied to human DNA polymorphism such as single nucleotide polymorphism13,28,32, short tandem repetition26, and mitochondrial DNA analsis16,19. Micro RNA was also analyzed via capillary LC/ESI-MS44. These published applications can also be realized by the present method. In addition, this method may be suitable for monitoring the interaction between oligonucleotide and low-molecular-weight compounds, such as the interaction of antibiotics and ribosomal RNA45 owing to the low-temperature separation. In such cases, oligonucleotides and low-molecular-weight compounds should be detected simultaneously, which is an advantage of using LC/ESI-MS.
There is a limitation that the instrumentation is not suitable for performing parallel analysis as in conventional techniques such as Sanger sequencing and real-time PCR. Additionally, the present method identifies only base composition and any base substitution within the same molecule cannot be distinguished. However, the MS-based DNA analysis described here may still have merit in terms of accuracy in comparison with the DNA binding dye-based techniques such as gel electrophoresis and real-time PCR.
The authors have nothing to disclose.
This work was supported by a Grant-in-Aid for Scientific Research by the Japanese Society for the Promotion of Science (15K08060).
|DNeasy Blood & Tissue Kit (50)
|DNA extraction kit
|Ethanol (99.5+ vol%)
|For dilution of wash buffers
|TE buffer (pH 8.0)
|For dilution of DNA sample
|Purified with reverse-phase cartridge column by the supplier
|Purified by HPLC by the supplier
|Generated with a Milli-Q Direct water purification system (Merck Millipore), used for sample preparation
|Detergent Free 5X Phusion HF Buffer
|Use instead of the provided buffer of DNA polymerase
|Pfu-X DNA polymerase
|dNTP mix (20 mM each)
|Supplied with the DNA polymerase
|10× Universal buffer M
|Containing 100 mM Tris-HCl (pH 7.5), 100 mM MgCl2, 10 mM dithiothreitol and 500 mM NaCl
|Eluent additive for LC-MS grade.
|Eluent additive for LC-MS grade.
|For preparation of calibrant.
|Sodium hydroxide (1.0 M)
|Dilute to 10 mM with ultrapurified water and use for titration of trifluoroacetic acid.
|For preparation of calibrant, LC/MS grade.
|For mobile phase, LC/MS grade.
|For mobile phase, LC/MS grade.
|Microcentrifuge tube (0.5 ml)
|PCR clean grade
|Microcentrifuge tube (1.5 ml)
|PCR clean grade
|Disposable UV cuvette.
|Fluorescent DNA stain
|Cosmospin filter G (0.2 μm)
|Made of hydrophilic polytetrafluoroethylene (PTFE) membrane. Any centrifugal filter unit (pore size, 0.2–0.5 μm) made of hydrophilic PTFE or another low-binding membrane is applicable.
|300-μl PP screw vial
|American Chromatography Supplies
|Preassembled screw cap and septa
|Monobis C18 analytical column (2.0×50 mm, mesopore size = 30 nm)
|Outside Japan, available for purchase from GL Sciences via its distributors. (http://www.glsciences.com/distributors/)
|Monobis C18 guard column
|Cadenza CW-C18 (3.0×250 mm)
|C18-bonded particulate silica column
|ProSwift RP-4H (1.0×250 mm)
|Poly(styrene-divinylbenzene) monolith column
|Themo Mixer C
|For digestion of fish tissues.
|Quantification of DNA concentration.
|For aqueous mobile phase to avoid evaporation of HFIP. This can be replaced by an ice box.
|Ultimate 3000 liquid chromatograph
|Q Exactive mass spectrometer
|Fourier transform mass spectrometer equipped with the modified Kingdon trap analyzer.
|Protein deconvolution 3.0
|Use version 3.0 or higher having an isotopic patter model of nucleotide.
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