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Figure 15.14. Determination of peptide sequence using nanoelectrospray ionization, and a very high-resolution mass analyzer (Q-TOF). In the rst quadrupole, a doubly charged peptide ion of m/z 625.41 was selected and later fragmented. The m/z CID spectrum yields the FGDYGSIDYGR sequence, shown at the top.23 [Reprinted, with permission, from E. Gustafsson, K. Thoren, T. Larsson, P. Davidsson, K. Karlsson, and C. L. Nilsson, Identi cation of Proteins from Escherichia coli Using Two-Dimensional Semi-Preparative Electrophoresis and Mass Spectrometry. Rapid Communications in Mass Spectrometry 15, 2001, 428 432. Copyright # 2001 John Wiley & Sons, Ltd.]
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is expected to improve as more experimental data are incorporated. In this regard, it must be noted that two mass spectra are comparable only if they were obtained under very similar instrumental/operational conditions; therefore, several different databases are needed, at least one for each ionization system. MALDI and ESI spectra are quite different, because these two ionization methods produce different types of ions. 15.7. NUCLEIC ACID APPLICATIONS The application of MS techniques to nucleic acids has been limited in comparison with protein polypeptide applications, mainly because of the dif culty of ionizing DNA or RNA, since their chemical and structural properties (mainly the negatively
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charged sugar-phosphate backbone) prevent ef cient positive ionization by MALDI or ESI. The use of NH counterions is an established method that allows the pro4 duction of oligonucleotide ions, but the sensitivity and resolution obtained is low. In general, the MALDI ionization process is very inef cient for nucleic acids in comparison with peptides. Generally, 100 times more DNA has to be used to achive similar signal intensity. Another approach being used to enhance the ionization process is the chemical modi cation of the nucleic acid molecule in order to improve ionic volatility. For example, the replacement of phosphate protons from native DNA backbones by alkyl groups,25 or the replacement of phosphate groups by phosphorothioate groups followed by alkylation26 have been reported. Another useful possibility is to use ESI in the negative ion mode, which yields a better signal for nucleotides. Multiply charged ions of the type M nH n are formed. Precise mass measurements for synthetic oligonucleotides with as many as 132 residues have been reported.27 Almost all procedures described for nucleic acid analysis (usually DNA) rely on PCR ampli cation. Short oligonucleotides have been sequenced (usually up to 30 nucleotides), although today MS is used more as a sequence con rmation method than as a sequence determination method. A nomenclature for ion fragments with higher probability of formation has been proposed,28 and some recent work describes procedures for DNA sequence elucidation using ESI ionization and ion fragmentation (CID spectra).29 The theory is similar to that used for protein sequencing; when a complete series of fragments is found, the mass difference between two consecutive fragments corresponds to a nucleotide residue mass. In practice, however, MS nucleic acid sequencing methodology is not competitive with alternative sequencing methods. Several assays have been patented for mutation detection; these are mainly designed for single-nucleotide polymorphism (SNP) analysis and use MALDI TOF spectrometry (InvaderTM, Sequazyme-PinPointTM assay, MassARRAYTM, GOODTM assay) all of which use PCR ampli cation, or require a high DNA concentration in the sample (InvaderTM). MAGIChipTM is a generic microchip-size device, where spots of polyacrylamide gel are separated by hydrophobic glass. Several gel squares (100 mm) are contained on one glass support, and are separated by 200 mm. Short DNA sequences are immobilized on the gel and are assayed for hybridization; later, the matrix compound is added, allowed to dehydrate, and the spots are analyzed by MALDI TOF. With this device, different SNP sequences are recognized by their different masses. Short oligonucleotide mass analysis (SOMA) assay uses PCR ampli cation followed by restriction endonuclease incubation to digest the ampli ed DNA; the short oligonucleotides are later separated by HPLC and analyzed by ESI Triple quadrupole MS (used in the negative ion mode), generating a ngerprint. This method has been used to genotype several variant sites in the human adenomatous polyposis coli gene, for which some variants have been associated with an increased risk of colorectal cancer.30
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