Sequence information, from electrospray spectrum

The TIC trace from this analysis, shown in Figure 5.5, exhibits a maximum at ca. 19 min, and a representative electrospray spectrum is illustrated in Figure 5.6. Transformation of the latter produces the spectrum presented in Figure 5.7 which indicates the presence of two species with relative molecular masses (RMMs) of 56 548.5 and 58 161.4 Da. These masses are lower than the value of 59.1 kDa calculated from previously obtained sequence information. 

Figure 2.5. Tandem mass spectrometry. A. A peptide mixture is electrosprayed into the mass spectrometer. Individual peptides from the mixture are isolated (circled peptide) and fragmented. B. The fragments from the peptide are mass analyzed to obtain sequence information. The fragments obtained are derived from the N or C terminus of the peptide and are designated "b" or "y" ions, respectively. The spectrum shown indicates peptides that differ in size by the amino acids shown.

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

More extensive structural information is obtained when using MS-MS, where information on the peptide sequence can be extracted from the fragmentation pattern. So far, CE-MS-MS has predominantly been performed with electrospray ionization (ESI)-triple quadrupole and ESI-ion trap instruments, as reviewed in Ref. 13. The advent of ESI-TOF and ESI-quadru-pole-TOF instruments is believed to have a strong impact on CE-MS. TOF instruments require an extremely short time to produce a full mass spectrum and are especially attractive as a detection device for a separation technique producing sharp peaks, as illustrated by the separation of three enkephalins in a time window of 6 s with detection by means of ESI-TOF [14],... 

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