Biomolecules mass spectra

More complex detective work is required to analyze large biomolecules and drugs. However, fragmentation generally follows predictable patterns, and one compound can be identified by comparing its mass spectrum with those of other known compounds with similar structures. In Fig. 2, we see the spectrum of a sample of blood from a newborn infant. The blood is being analyzed to determine whether the child has phenylketonuria. The presence of the compound phenylalanine is a positive indication of the condition. Some... 

The mass spectrum produced should provide unambiguous molecular weight information from the wide range of compounds amenable to analysis by HPLC, including biomolecules with molecular weights in excess of 1000 Da. The study of these types of molecule by mass spectrometry may be subject to limitations associated with their ionization and detection and the mass range of the instrument being used. 

ESI-MS has emerged as a powerful technique for the characterization of biomolecules, and is the most versatile ionization technique in existence today. This highly sensitive and soft ionization technique allows mass spectrometric analysis of thermolabile, non-volatile, and polar compounds and produces intact ions from large and complex species in solution. In addition, it has the ability to introduce liquid samples to a mass detector with minimum manipulation. Volatile acids (such as formic acid and acetic acid) are often added to the mobile phase as well to protonate anthocyanins. A chromatogram with only the base peak for every mass spectrum provides more readily interpretable data because of fewer interference peaks. Cleaner mass spectra are achieved if anthocyanins are isolated from other phenolics by the use of C18 solid phase purification. - ... 

After gas-phase ions have been generated, several approaches may be used to determine their mass. In time of flight (TOF) analysis, the ions are accelerated in an electric field toward a detector (Figure 3.40). The lighter ions are accelerated more, travel faster, and arrive at the detector first. Tiny amounts of biomolecules, as small as a few picomoles (pmol) to femtomoles (fmol), can be analyzed in this manner. A MALDI-TOF mass spectrum for a mixture of the proteins insulin and p-lactoglobulin is shown in Figure. 1.41. The masses determined by MALDI-TOF are 5733.9 and 18,364,... 

Various forms of tandem mass spectroscopy (MS/MS) have also been used in the analysis of biomolecules. Such instruments consist of an ionisation source (ESI or MALDI or other) attached to a first mass analyser followed by a gas-phase collision cell. This collison cell further fragments the selected ions and feeds these ions to a second mass detector. The final mass spectrum represents a ladder of fragment ions. In the case of peptides the collision cell usually cleaves the peptides at the amide bond. The ladder of resulting peptides reveals the sequence directly [496]. Thus, tandem MS instruments, such as the triple quadrupole and ion-trap instruments have been routinely applied in LC-MS/MS or ESI-MS/MS for peptide sequencing and protein identification via database searching. New configurations, which have been moving into this area include the hybrid Q-TOF [498], the MALDI-TOF-TOF [499] and the Fourier transform ion cyclotron resonance instruments [500]. 

FIGURE 3.39 MS/MS enables the molecular identification of interested ions directly on the tissue surface. A. Product ion mass spectrum on the liver section of m/z 725. The NL of 59 and 124 u observed in the spectra is trimethylamine and cyclophosphate, indicating phosphocholine structure. This fragmentation occurred when alkah metal adducted to the precursor ion. The biomolecule of m/z 725 was suggested to be the sodiated molecule of SM(16 0). B. Product ion mass spectra on the Uver section of m/z 616.2 (a) and 557.2 (b).The m/z value and fragment patterns indicate that the product ion of m/z 616 is heme B. Consecutive NLs of 73, 59, and 45 Da correspond to CH2CH2COOH, CH2COOH, and COOH, respectively. The molecular structure of heme B is shown as an inset in (a). 

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