Passage
Electrospray ionization mass spectrometry (ESI-MS) employs a soft ionization technique that, unlike traditional ionization via electron bombardment, results in little to no fragmentation of target molecules. During electron bombardment, electrons collide with the analyte and cause the molecule to lose an electron whereas during ESI-MS, ions are produced by passing the analyte solution through an electrospray needle that has a potential difference applied with respect to a counterelectrode. This leads to the formation of charged droplets, which are repelled from the needle toward the counterelectrode and mass spectrometer, as shown in Figure 1.
Figure 1 ESI-MS ionization mechanismDuring the droplet's flight, the solvent is evaporated off analyte molecules. Ionization occurs through the protonation of basic sites on target molecules. Reaction 1 shows the number of protons n added to molecule M.M → (M + nH) ⁿ⁺Reaction 1Researchers used ESI-MS to quantify the formation of four pyridyloxobutyl-DNA (POB-DNA) adducts, shown in Figure 2. POB-DNA adduct formation involves highly mutagenic pyridyloxobutylating agents, which are formed from cytochrome P450-mediated hydroxylation of 4-(methylnitrosamino) -1-(3-pyridyl) -1-butanone (NNK) , an abundant N-nitrosamine carcinogen found in cigarette smoke. Adducts were produced in vitro by treating samples of calf thymus DNA with NNK. The DNA adducts were then separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and analyzed via ESI-MS.
Figure 2 Molecular structure of POB-DNA adducts
Adapted from Lao Y, Villalta PW, Sturla SJ, Wang M, Hecht SS. Quantitation of pyridyloxobutyl DNA adducts of tobacco-specific nitrosamines in rat tissue DNA by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Chem Res Toxicol. 2006.
-Prior to adduct formation, DNA was isolated from a cellular mixture. Membrane lipids were first extracted using an organic solvent, which was then evaporated, and DNA was precipitated from the aqueous layer using cold ethanol and sodium acetate. Which of the following mechanisms allowed for DNA precipitation?

Question

Passage
Electrospray ionization mass spectrometry (ESI-MS) employs a soft ionization technique that, unlike traditional ionization via electron bombardment, results in little to no fragmentation of target molecules. During electron bombardment, electrons collide with the analyte and cause the molecule to lose an electron whereas during ESI-MS, ions are produced by passing the analyte solution through an electrospray needle that has a potential difference applied with respect to a counterelectrode. This leads to the formation of charged droplets, which are repelled from the needle toward the counterelectrode and mass spectrometer, as shown in Figure 1.

Figure 1 ESI-MS ionization mechanismDuring the droplet's flight, the solvent is evaporated off analyte molecules. Ionization occurs through the protonation of basic sites on target molecules. Reaction 1 shows the number of protons n added to molecule M.M → (M + nH) ⁿ⁺Reaction 1Researchers used ESI-MS to quantify the formation of four pyridyloxobutyl-DNA (POB-DNA) adducts, shown in Figure 2. POB-DNA adduct formation involves highly mutagenic pyridyloxobutylating agents, which are formed from cytochrome P450-mediated hydroxylation of 4-(methylnitrosamino) -1-(3-pyridyl) -1-butanone (NNK) , an abundant N-nitrosamine carcinogen found in cigarette smoke. Adducts were produced in vitro by treating samples of calf thymus DNA with NNK. The DNA adducts were then separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and analyzed via ESI-MS.

Figure 2 Molecular structure of POB-DNA adducts
Adapted from Lao Y, Villalta PW, Sturla SJ, Wang M, Hecht SS. Quantitation of pyridyloxobutyl DNA adducts of tobacco-specific nitrosamines in rat tissue DNA by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Chem Res Toxicol. 2006.
-Prior to adduct formation, DNA was isolated from a cellular mixture. Membrane lipids were first extracted using an organic solvent, which was then evaporated, and DNA was precipitated from the aqueous layer using cold ethanol and sodium acetate. Which of the following mechanisms allowed for DNA precipitation?

Quizplus · Accepted Answer

11ecba2d_127f_74b0_a3bf_b9b135eee9fd_MD0008_00 "Like dissolves like" is the driving principle for extraction procedures. During extraction, two immiscible solvents (solvents that will not remain mixed) can be used to separate solutes (dissolved particles) according to differences in polarity. This procedure often entails the addition of an organic solvent to an aqueous mixture. After gentle shaking, hydrophobic solutes such as lipids separate into the organic layer, and polar or charged solutes such as nucleic acids remain in the aqueous layer. During extraction procedures, a molecule's charge or polarity may be modified to increase or decrease its affinity for a particular solvent. In this example, DNA remains in aqueous solution due to negatively charged phosphate groups (PO₃⁻) on the sugar phosphate backbone. To precipitate the DNA from aqueous solution, its charge must be neutralized through extraction with ethanol and a salt such as sodium acetate. Gentle mixing with ethanol disrupts the hydration shell around DNA molecules. Sodium cations then neutralize DNA's charge via ionic bonding with phosphate groups, making DNA less hydrophilic, decreasing its affinity for the aqueous solvent, and allowing it to precipitate more efficiently. (Choice A) Sodium acetate, the sodium salt of acetic acid, is a weak base. The addition of sodium acetate to water increases the solution's pH instead of reducing it. (Choice B) Ethanol has a lower polarity than water. The addition of ethanol disrupts the solvation shell surrounding DNA, allowing for sodium cations to bond with DNA. (Choice C) Cold ethanol is used because it is less likely than warm ethanol to disrupt DNA hydrogen bonds. However, the solution is not cold enough to freeze DNA. Educational objective: "Like dissolves like" is the driving principle for extraction procedures. During extraction, nonpolar solutes separate into the organic (nonpolar) layer whereas polar solutes separate into the aqueous (polar) layer. Modifying a molecule's charge or polarity modifies its affinity for each layer.

Passage Electrospray Ionization Mass Spectrometry (ESI-MS) Employs a Soft Ionization Technique