Peptide fragmentation spectrum

Figure 6.3. Real-life example of a tandem MS experiment in an electrospray ion trap instrument. Top panel a complex peptide mixture. Middle panel ion at 1318.9 m/z was isolated from other sample components. Note the lack of any other peaks and a very low background. Bottom panel fragmentation spectrum of the selected parent ion (1318.9 m/z), note the different scale of the m/z axis. All peaks seen in this mass spectrum are product ions that were formed due to the controlled fragmentation of the parent ion. The main peak at 1300.8 m/z corresponds to the loss of water molecule, a lower intensity parent ion at 1318.9 m/z is also seen.

Figure 6.4. Fragmentation spectrum of a tryptic peptide obtained from bovine serum albumin. Peptide sequence LGEYGFQNALIVR, monoisotopic [M + H]+ = 1479.796, monoisotopic [M+2H]2+ =740.402. Upper panel full scan MS spectrum. Lower panel MS/MS spectrum of a doubly-charged ion at 740.7 m/z with a ladder of y ions, the distances between which correspond to amino acid residues (upper row of letters). A shorter series of b ions is also seen (lower row of letters). See Fig. 6.5 for description of nomenclature. Note the often observed phenomenon where multiply-charged ions lose the charge during fragmentation process and, therefore, have higher m/z values than the original parent ion.

Figure 6.14. Example 2 fragmentation spectrum of singly charged peptide, precursor mass of 1098.6.

Figure 6.19. Fragmentation spectrum of doubly charged peptide precursor at 741.0Th, used in Example 3.

Fragmentation of peptides can also be observed with FTICR instruments. Infrared multiple photon dissociation (IRMPD) and electron capture dissociation (ECD) have been introduced as two alternative dissociation methods to the low-energy CID method. The IRMPD method produces many fragments that make the spectrum very complex and difficult to interpret. Some of the fragment types observed with IRMPD are b and y type ions or these ions that have lost ammonia or water. However, most of them are not these types of fragment ions. 

ECD has recently been introduced as an alternative activation method to obtain fragmentation of multiply protonated peptides [57]. An example of an ECD fragmentation spectrum... 

Interpretation of the spectra is based on the mechanisms and the fragmentation pathways described above, as shown by the following example. A CID MS/MS fragment spectrum of a peptide with sequence Gly-Ile-Pro-Thr-Leu-Leu-Leu-Phe-Lys measured at high energy is shown in Figure 8.13. This spectrum contains the complete series of b ions, thus allowing one to deduce the peptide sequence from the N-terminal acid to the C-terminal acid, whereas the series of y ions allows identification of the sequence in the reverse direction. In fact, the mass difference of 97 Da between peak b2 and b3 indicates that the amino acid in position 3 corresponds to a proline (see Table 8.2). Similarly, the 147 Da difference between peaks yi and y2 indicates that, the amino acid in the next-to-last position is a phenylalanine. The m/z values of ions W3, W4, W5 and wg imply that the amino acid in... 

D Fragment spectrum of the phosphorylated peptide ApTSNVFAMFDQSQIQEFK or ATpSNVFAMFDQSQIQEFK, respectively. This peptide contained three possible phosphorylation sites (Thr-2, Ser-3, and Ser-12). The comparison of the two spectra revealed Thr-2 or Ser-3 as phosphorylated residues. As no secession of-98 Da was observed till y,j a phosphorylation of Ser-13 could be excluded. On the basis of the MS/MS spectrum the exact site of phosphorylation was not determinable. 

Fidder et al. introduced an electrospray-ionization tandem mass spectrometry method for diagnosing OP exposure by measuring the mass of the OP-labeled active site peptide of human butyrylcholinesterase (Fidder et al, 2002). His starting material was 0.5 ml of human plasma from a victim of the Tokyo subway attack. The mass of the active site peptide was higher by 120 atomic mass units, compared to the mass of the unlabeled active site peptide. This added mass was exactly the added mass expected from sarin. The peptide s MS-MS fragmentation spectrum yielded the sequence of the peptide, and verified that the OP label was on serine 198, the active site serine. Examples of the MS-MS spectra from tryptic peptides of pure, OP-labeled human butyrylcholinesterase are shown in Figure 56.1. 

Fig. 4. HPLC is used to separate the components of a protein digest mixture, (a) Base peak ion current as a function of time. MS and MS/MS mass spectra are recorded in real time, (b) Full MS spectrum obtained at retention time = 26.47 min. Two main coeluting components are detected (see, e.g., doubly charged ions at miz 571.4 and 643.2). (c) The tandem MS/MS (fragmentation) spectrum of the doubly charged peptide ion at m/z 571. The mIz values of the fragments are used to sequence the peptide. 

Fig. 1 is an illustration of the need for de novo sequencing. The portion of the protein sequence shown in the box corresponds to the mass of a peptide produced by proteolysis. Above this sequence is shown a series of three amino acid residues that have been found to be present in a fragmentation spectrum, a sequence tag [4]. Note that, in general, the order of the residues found is not known, i.e., they could... 

An alternative approach to peptide sequencing uses a dry method in which the whole sequence is obtained from a mass spectrum, thereby obviating the need for multiple reactions. Mass spec-trometrically, a chain of amino acids breaks down predominantly through cleavage of the amide bonds, similar to the result of chemical hydrolysis. From the mass spectrum, identification of the molecular ion, which gives the total molecular mass, followed by examination of the spectrum for characteristic fragment ions representing successive amino acid residues allows the sequence to be read off in the most favorable cases. 

Figure 2.3. A. Mass spectrometer consisting of an ionization source, a mass analyzer and an ion detector. The mass analyzer shown is a time-of -flight (TOF) mass spectrometer. Mass-to-charge (m/z) ratios are determined hy measuring the amount of time it takes an ion to reach the detector. B. Tandem mass spectrometer consisting of an ion source, a first mass analyzer, a collision cell, a second mass analyzer and a detector. The first mass analyzer is used to choose a particular peptide ion to send to the collision cell where the peptide is fragmented. The mass of the spectrum of fragments is determined in the second mass analyzer and is diagnostic of the amino acid sequence of the peptide. Figure adapted from Yates III (2000).

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.

Figure 12.4 MALDI analysis of peptides prepared in situ from a 10 1 mixture of Bacillus cereus and Bacillus anthracis sp. Sterne.82 (a) Survey spectrum of peptide products. Species assignments are indicated on the figure. (b) Spectrum of fragment ions produced by low-energy collisions of the Bacillus cereus-specific peptide of mass 1529. (c) Spectrum of fragment ions produced by low-energy collisions of ions of the Bacillus anthracis peptide of mass 1528.

MS/MS Duty Cycle Typical MS/MS analysis is a serial process, relying on the selection of precursors (peptides) in MS mode, followed by high-energy fragmentation in MS/MS. This process is termed data dependent acquisition (DDA). The duty cycle for the completion of MS and MS/MS cycles (the time necessary for MS/MS spectrum acquisition) is of primary importance. When the separation performance is viewed from the mass spectrometry perspective, the peak capacity can be characterized by the number of MS/MS scans, yielding successful... 

---