Nucleotides, mass differences

Figure 10.3. Mass array, (a) Primer binding (b) primer extension enzyme, ddATP and dCTP/ dGTP/dTTP addition (c) primer terminates (d) primer extension products ready for MALDI-MS (e) MS spectrum of primer extension products. Each addition of a nucleotide to the primer extension product increases the mass by 289 to 329 Da, depending on the nucleotide added. The mass difference is easily resolved by MALDI-TOF, which has the ability to detect differences as small as 3 Da. Printed by kind permission of Sequenom. (See color insert.)...

Another important advantage of mass spectrometry is its capacity to verify the incorporation of modified nucleotides. Figure 8.31 displays the mass spectrum of a synthetic oligonucleotide whose theoretical mass is 5838 Da. In addition to the desired nucleotide, other compounds are present. The mass difference between the peaks (111-151 Da) corresponds to the masses of the nucleic bases. This suggests that depurination reactions have occurred during the synthesis. 

The structure of RNA differs from that of DNA in several respects. First, as shown in Fignre 25.17, the fonr bases fonnd in RNA molecules are adenine, cytosine, guanine, and nracil. Second, RNA contains the sugar ribose rather than the 2-deoxyribose of DNA. Third, chemical analysis shows that the composition of RNA does not obey Chargaff s rnles. In other words, the pnrine-to-pyrimidine ratio is not equal to 1 as in the case of DNA. This and other evidence rule out a double-heUcal structure. In fact, the RNA molecnle exists as a single-strand polynucleotide. There are actually three types of RNA molecules—messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (fRNA). These RNAs have similar nucleotides but differ from one another in molar mass, overall strnctnre, and biological functions. 

We have successfully used MALDI-TOF mass spectrometry to identify stacked 5-mers during CSH [10], Several samples of DNA were hybridised to different microchip-immobilised oligonucleotides in the presence of a 5-mer mixture. The molecular-mass differences among any of the four nucleotides — dAp (313.2 Da), dCp (289.2 Da), dGp (329.2 Da) and dTp (304.2 Da) — constitute at least 9 Da, which is easily resolved by using MALDI-TOF. 

A simple way to avoid ambiguities related to the inconvenient mass difference between pinpoint products is the use of mass-modified terminator nucleotides, as introduced by Smith and coworkers (153). The rationale design of terminator nucleotides with a perfect mass separation can be achieved by selecting appropriate mass-modifying chemical moieties. 

A second advantage of the LC-ESI-MS approach is the increased mass accn-racy of the measurement, making possible the detection of small mass differences between proteins. These mass differences can be eqirivalent to single-nucleotide polymorphisms (SNP) mutations or post-translational modifications. Recent work by McFarland and coworkers (2014) best illustrates the implementation of LC-(ESI)-MS (intact protein mass) and MS/MS top-down analyses to bacteria differentiation at the strain level. Proteins extracted from bacterial samples of Salmonella typhimurium (strain LT2) and S. heidelberg (strain A3 9) were first separated by reversed-phase (RP) LC and the eluent analyzed directly by ESI-MS using Q-TOF MS system (operated in the full-scan mode or MS). Following the data processing... 

The utility of any mass spectrometric sequencing method that relies on consecutive backbone cleavages depends on the formation of a mass ladder. The sequence information is obtained by determining the mass difference between successive peaks in the mass spectrum. In the case of oligodeoxynucleotides, the expected mass differences between successive peaks will correspond to the loss of dC = 289.25 u, dT = 304.26 u, dA = 313.27 u and dG = 329.27 u (all values are atomic mass-based). With oligoribo-nucleotides, the mass differences will be rC = 305.25 u, rU = 306.26 u, rA = 329.27 u and rG = 345.27 u (all values are atomic mass-based). 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

A modified nucleotide found in RNA sequencing could either be a new nucleotide of unknown chemical structure or it could correspond to an already known modified nucleotide (up to now about 90 different modified nucleotides have been identified in RNA). Keith [124] proposed preparative purifications of major and modified ribonucleotides on cellulose plates, allowing for their further analysis by UV or mass spectrometry. Separation was realized by two-dimensional elution using the following mobile phases (1) isobutyric acid-25% ammonia-water (50 1.1 28.9,... 

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