High-Performance Liquid Chromatography−Electrospray Ionization Mass Spectrometry of Single- and Double-Stranded Nucleic Acids Using Monolithic Capillary Columns

Ion-pair reversed-phase high-performance liquid chromatography online hyphenated to electrospray ionization mass spectrometry (ICEMS) represents an efficient method for the characterization of nucleic acids amplified by polymerase chain reaction (PCR). Since sample preparation is limited to PCR, the optimization of its solution conditions is of utmost importance for efficient mass spectrometric detection. The compatibility of a number of different commercially available PCR components including DNA polymerases, deoxynucleotide triphosphates, bovine serum albumin, enhancer, and ionic buffers was evaluated. These experiments revealed that higher concentration of enhancer and detergents such as Tween-20 or Nonidet P-40 impairs the mass spectrometric detection of nucleic acids and should be avoided within the PCR mixture. The optimized analytical platform was applied to the characterization of PCR products covering parts of the first hypervariable region of the noncoding mitochondrial control region. Truncated amplicons were detected attributable to the use of low quality primers. Furthermore, due to the proofreading activity of the applied polymerase system, mismatches between the primer and the target sequence located at the last or the second last base at the 3=-end of primers were corrected and detected within the corresponding amplicons. (J Am Soc Mass Spectrom 2006, 17, 124 -129)

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“…The 60 ϫ 0.2 mm i.d. monolithic capillary column was prepared according to the published protocol [12].…”

Section: Methodsmentioning

confidence: 99%

Some guidelines for the analysis of genomic DNA by PCR-LC-ESI-MS

Oberacher

Niederstätter

Casetta³

et al. 2006

J. Am. Soc. Mass Spectrom.

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“…The 60 ϫ 0.2 mm i.d. monolithic capillary column was prepared according to the published protocol [13] and connected directly to the spray capillary (fused silica, 90 m o.d., 20 m i.d., Polymicro Technologies, Phoenix, AZ) by means of a microtight union (Upchurch Scientific, Oak Harbor, WA). A syringe pump equipped with a 250 L glass syringe (Unimetrics, Shorewood, IL) was used for the post-column addition of a 3.0 L/min flow of acetonitrile by means of a tee-piece.…”

Section: Capillary Hplc Coupled To Esi-msmentioning

confidence: 99%

“…In this context, we have introduced ion-pair reversed-phase high-performance liquid chromatography on-line interfaced to electrospray ionization mass spectrometry (ICEMS) as an analytical tool for the charac-terization of DNA sequences chain reaction (PCR) [13][14][15][16][17][18]. ICEMS enables the detection of SNPs based on the determination of the intact molecular mass using low femtomol amounts of DNA fragments up to lengths of more than 100 base pairs.…”

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Applicability of tandem mass spectrometry to the automated comparative sequencing of long-chain oligonucleotides

Oberacher¹,

Parson²,

Oefner³

et al. 2004

J. Am. Soc. Mass Spectrom.

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An algorithm for the comparative sequencing (COMPAS) of oligonucleotides is shown to be suitable for the sequence verification of nucleic acids ranging in length from a few to 80 nucleotides. The algorithm is based on the matching of a fragment ion spectrum generated by collision-induced dissociation to m/z values predicted from a known reference sequence employing established fragmentation pathways. Prior to mass spectrometric investigation, the oligonucleotides were on-line purified by ion-pair reversed-phase high-performance liquid chromatography using monolithic separation columns. This study evaluated the potential and the limits of COMPAS regarding the length and the charge state of oligonucleotides, the selected collision energy, and the analyzed amount of sample using a quadrupole ion trap mass spectrometer. The results revealed that oligonucleotides could be very reliably resequenced up to 60-mers, although the algorithm was successfully used to even verify sequences up to 80-mers. The relative collision energy was typically in the range between 13 and 20%, which allowed in most cases a verification of the reference sequence in a window of at least three consecutive collision energies. To select a proper charge state for fragmentation, a compromise had to be found between high signal intensity and low charge state. Furthermore, by reducing the eluent flow rate during elution of the oligonucleotide, the sequence of a 50-mer was successfully verified from the analysis of 295 fmol of the raw product. COMPAS was proven to be reproducible and was applied to the genotyping of the polymorphic, Y-chromosomal locus M9 contained in a 62-base pair polymerase chain reaction product. (J Am Soc Mass Spectrom 2004, 15, 510 -522)

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“…Consequently, MSMS of oligodeoxynucleotides has been successfully applied to characterize or confirm structures [11], to detect chemical modifications [2,10,18], and to identify sequence variations [14, 19 -21]. However, the deduction of sequence information is time-consuming, highly technical, lacking strict objectivity due to reliance on human interpretation, and can be performed only in laboratories with extensive experience in MSMS.…”

mentioning

confidence: 99%

“…In this context the combination of ion-pair reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry (ICEMS) has emerged as a versatile tool for the analysis of single-and double-stranded nucleic acids ranging in size from a few nucleotides (nt) up to several hundred base pairs (bp) [2][3][4]. Although the occurrence of a sequence variation is predictable on the basis of molecular mass measurements [5][6][7], detailed sequencing information can only be obtained from selective decomposition of oligonucleotides by tandem mass spectrometry (MSMS).…”

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confidence: 99%

Automated de novo sequencing of nucleic acids by liquid chromatography-tandem mass spectrometry

Oberacher

Mayr

Huber

2004

J. Am. Soc. Mass Spectrom.

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We present the first global computer-aided sequencing algorithm for the de novo determination of short nucleic acid sequences. The method compares the fragment ion spectra generated by collision-induced dissociation of multiply charged oligodeoxynucleotide-ions to the m/z values predicted employing established fragmentation pathways from a known reference sequence. The closeness of matching between the measured spectrum and the predicted set of fragment ions is characterized by the fitness, which takes into account the difference between measured and predicted m/z values, the intensity of the fragment ions, the number of fragments assigned, and the number of nucleotide positions not covered by fragment ions in the experimental spectrum. Smaller values for the fitness indicate a closer match between the measured spectrum and predicted m/z values. In order to find the sequence most closely matching the experimental spectrum, starting from a given nucleotide composition all possible oligonucleotide sequences are assembled followed by identification of the correct sequence by the lowest fitness value. Using this concept, sequences of 5-to 12-mer oligodeoxynucleotides were successfully de novo determined. High sequence coverage with fragment ions was essential for obtaining unequivocal sequencing results. Moreover, the collision energy was shown to have an impact on the interpretability of tandem mass spectra by the de novo sequencing algorithm. Experiments revealed that the optimal collision energy should be set to a value just sufficient for complete fragmentation of the precursor ion. T he hyphenation of liquid chromatography (LC) to mass spectrometry (MS) is one of the most powerful methods for the characterization of oligonucleotides today [1]. In this context the combination of ion-pair reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry (ICEMS) has emerged as a versatile tool for the analysis of single-and double-stranded nucleic acids ranging in size from a few nucleotides (nt) up to several hundred base pairs (bp) [2][3][4]. Although the occurrence of a sequence variation is predictable on the basis of molecular mass measurements [5][6][7], detailed sequencing information can only be obtained from selective decomposition of oligonucleotides by tandem mass spectrometry (MSMS).The principles of gas-phase collision-induced dissociation (CID) reactions of oligonucleotides have been studied extensively [8 -17] and the resulting product ion spectra are predictable on the basis of known fragmentation pathways. Consequently, MSMS of oligodeoxynucleotides has been successfully applied to characterize or confirm structures [11], to detect chemical modifications [2,10,18], and to identify sequence variations [14, 19 -21]. However, the deduction of sequence information is time-consuming, highly technical, lacking strict objectivity due to reliance on human interpretation, and can be performed only in laboratories with extensive experience in MSMS. Hence, automation of pro...

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High-Performance Liquid Chromatography−Electrospray Ionization Mass Spectrometry of Single- and Double-Stranded Nucleic Acids Using Monolithic Capillary Columns

Cited by 277 publications

References 41 publications

Some guidelines for the analysis of genomic DNA by PCR-LC-ESI-MS

Some guidelines for the analysis of genomic DNA by PCR-LC-ESI-MS

Applicability of tandem mass spectrometry to the automated comparative sequencing of long-chain oligonucleotides

Automated de novo sequencing of nucleic acids by liquid chromatography-tandem mass spectrometry

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