Characterization of synthetic nucleic acids by electrospray ionization quadrupole time‐of‐flight mass spectrometry

Abstract

The potential of electrospray ionization quadrupole-quadrupole-time-of-flight mass spectrometry (ESI-QqTOF-MS) for the characterization of synthetic nucleic acids was evaluated. Oligonucleotides ranging in size from 12 up to 51 nucleotides were analyzed via direct infusion MS as well as via liquid chromatography (LC) online hyphenated to MS. These experiments proved the outstanding mass spectrometric performance of the TOF mass analyzer in regard of accuracy, reproducibility, resolution, and sensitivity. During a 1-min run, the monoisotopic mass of (dT)(24) was measured with a maximum relative mass deviation of 7.64 ppm proving the high mass accuracy of the TOF analyzer. Over a period of 1 h, mean deviations were determined in the range between -3.58 ppm and 3.06 ppm demonstrating the high stability of the applied external calibration. The molecular mass of a 51-mer was measured with a deviation smaller than 3.23 ppm from the theoretical value. The resolution exceeded a value of m/Deltam = 20 000 (m is the measured mass and Deltam the full peak width at half-maximum), which enabled the separation of the isotopic peaks of all investigated oligonucleotides. Because of the outstanding transmission and detection efficiency of the TOF mass analyzer, detection limits in the amol/microl to low fmol/microl range were reached. The usability of LC-ESI-QqTOF-MS for the qualitative and quantitative analysis of synthetic oligonucleotide mixtures was demonstrated.

Orbitrap mass spectrometry: Instrumentation, ion motion and applications

“…The QqTOF, which can be regarded as a triple quadrupole (QqQ) instrument where the third quadrupole is replaced by an orthogonal TOF, was originally used for rapid de novo peptide sequencing (Shevchenko et al, 1997) but has found application in many areas of research including metabolite (Levsen et al, 2005), nucleic acid (Oberacher, Niederstatter, & Parson, 2005) and glycoprotein (Morris et al, 2007) analysis. However, for scan-types in which a single ion is monitored such as precursor ion and neutral loss scans (important experiments for structural characterization), the sensitivity of the QqTOF (5-30% duty cycle) is lower than QqQ instruments because more ions are lost in TOF compared with a third quadrupole.…”

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

“…ESI-MS was performed on a QSTAR XL mass spectrometer (Applied Biosystems) equipped with a modified TurboIonSpray source [6,13]. The exit of the capillary column was connected directly to the transfer line by means of a microtight union (Upchurch Scientific, Oak Harbor, WA).…”

“…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 as previously described [6,13]. The spray voltage was typically in the range of 3.0 to 3.5 kV.…”

Increasing the discrimination power of forensic STR testing by employing high-performance mass spectrometry, as illustrated in indigenous South African and Central Asian populations

“…The sequences of the PCR primers were taken from [6]. All oligonucleotides were obtained from Sigma-Aldrich and quality-checked by ICEMS [10] before use in PCR.…”

“…The gradient was started 5 min after the sample injection event. Eluting nucleic acids were detected online by negative electrospray ionization-mass spectrometry which was performed on a QSTAR XL mass spectrometer (AB) equipped with a modified TurboIonSpray source [10,12]. Mass calibration and optimization of instrumental parameters was performed as described elsewhere [10,12].…”