title: Evaluation of the LTQ-Orbitrap mass spectrometer for the analysis of polymerase chain reaction products.
abstract: We have investigated the potential and robustness of the off-line coupling of polymerase chain reaction (PCR) with electrospray ionization mass spectrometry (ESI-MS), for further applications in the screening of single-nucleotide polymorphisms (SNPs). This was based on recently reported data demonstrating that anion-exchange solid-phase extraction was the most efficient technique for efficiently desalting PCR products, with a recovery of ∼70%. Results showed that this purification approach efficiently removes almost all the chemicals commonly added to PCR buffers. ESI-MS analysis of a model 114-bp PCR product performed on the LTQ-Orbitrap instrument demonstrated that detection limits in the nM range along with an average mass measurement uncertainty of 9.15 ± 7.11 ppm can be routinely obtained using an external calibration. The PCR/ESI-MS platform was able to detect just a few copies of a targeted oligonucleotide. However, it was shown that if two PCR products are present in a mixture in a ratio higher than 10 to 1, the lower abundance one might not be reproducibly detected. Applications to SNPs demonstrated that an LTQ-Orbitrap with a resolution of 30 000 (at m/z 400) easily identified a single (A ↔ G) switch, i.e. a 16 Da difference, in binary mixtures of ∼ 35 kDa PCR products. Complementary experiments also showed that the combination of endonucleases and ESI-MS could be used to confirm base composition and sequence, and thus to screen for unknown polymorphisms in specific sequences. For example, a single (T ↔ A) switch (9 Da mass difference) was successfully identified in a 114-bp PCR product.
authors.affiliation: CEA, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, 91191 Gif-sur-Yvette, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Manduzio
authors.forename: Eric
authors.initials: E
authors.lastname: Ezan
authors.forename: François
authors.initials: F
authors.lastname: Fenaille
mesh_terms: D001173:Arthrobacter / Q000235:genetics; D001483:Base Sequence; D002138:Calibration; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D007474:Ion Exchange; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D009841:Oligonucleotides; D009930:Organic Chemicals; D012336:RNA, Ribosomal, 16S; D019003:Deoxyribonuclease HpaII