Validation of DNA Sequences Using Mass Spectrometry Coupled with Nucleoside Mass Tagging

(A) Negative-ion MALDI-TOF-MS spectrum of unlabeled 10-bp PCR product after Hph I restriction digestion. The (−)-strand has an m/z ratio of 3068.2, and the (+)-strand has an m/z ratio of 3120.7. (B) Negative-ion MALDI-TOF-MS spectrum of 50% ¹³C/¹⁵N-dATP-labeled 10-bp product. Four different peaks represent the (−)-strand with m/z ratios of 3069.2, 3082.3, 3098.2, and 3112.1, reflecting the presence of three labeled adenines. Three different peaks with m/z ratios of 3120.6, 3135.8, and 3151.0 are detected for the (+)-strand. The peaks are separated by ∼15 Da each. (C) Negative-ion MALDI-TOF-MS spectrum of 50%¹³C/¹⁵N-dTTP-labeled 10-bp product. The (−)-strand has three peaks with m/z ratios of 3068.4, 3079.9, and 3091.7. The (+)-strand was represented with four peaks with m/z ratios of 3118.5, 3132.8, 3144.5, and 3156.9. (D) Negative-ion MALDI-TOF-MS spectrum of 50% ¹³C/¹⁵N-dCTP-labeled 10-bp product. The (−)-strand had four peaks with m/z ratios of 3070.3, 3081.8, 3093.3, and 3105.7, whereas the (+)-strand had only two peaks with an m/z ratio of 3121.4 and 3133.6. (E) Negative-ion MALDI-TOF-MS spectrum of 50% ¹³C/¹⁵N-dGTP-labeled 10-bp product. The (−)-strand displayed two peaks with m/z values of 3067.0 and 3081.8. The (+)-strand had four peaks with a mass of 3120.4, 3135.1, 3150.6, and 3164.0. “+Na⁺” indicates sodium adducts.