Sequence peak ions

Rg. 13. Molecular ion and sequence peak ions of an acyl tripeptide methylester... 

Figure 1.13 shows the spectrum of a hexapeptide [181] as its permethy-lated derivative with the ions indicating the amino acid order. The rapid decrease in the intensity of the important sequence-determining ions at higher mass is evident and a principal reason why MS often requires several times as much material as the common micro wet chemical methods for sequencing small peptides. The chemical ionization mass spectra of some N -acyl permethylated simple peptides show a much more even distribution of the sequencing peaks and hence require a lower sample level than for El spectra [187]. This may well prove of value in the future. Cl has also been applied directly to peptides [188] where up to six amino acids units have been introduced by the direct insertion probe. 

A direct investigation of unsubstituted A-imines by mass spectrometry is impossible since these compounds are stable only in solution. Unsubstituted pyridine A-imines are formed on heating A-aminopyridinium chlorides in the direct inlet system at 200°, as was shown by Tamura and co-workers.126 The mass spectrum of the unsubstituted A-imines (see Scheme 8) contains peaks due to the loss of 15 (NH) and 16 (NH2) mass units from the molecular ion in a ratio of 2.5 1. HCN is also eliminated from the molecular ion to a small extent, the reaction presumably proceeding via the sequence molecular ion — 85 —> 86 see also thermal A-imine diazepine tautomcrism in Section II, A,3. 

Fig. 11 shows the structure of permethylated MDP with the principal fragmentations (dotted lines) Fig. 12 shows the corresponding mass spectrum. The molecular ion peak is at m/e 618 and a sequence peak at m/e 417, accompanied by m/e 389 (loss of CO) and 357 (loss of MeOH). Loss of the entire pyranose ring gives the ion at m/e 342. The complementary C-terminal fragment to m/e 417 is at m/e 203. Peaks at m/e 260 and 228 (260-MeOH) contain the intact pyranose ring and are known to occur in the spectra of permethylated-N-acetyl hexosamines. In these fragments the entire lactyl peptide chain has been eliminated. 

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. 

Chromatographic peak Retention time (min) m/z Ion assigned Fragment and residue number Sequence... 

In our example spectrum (Fig. 6.11), the majority of most intense peaks belong to N-terminal fragments, leaving only low intensity peaks for potential C-terminal ions. In such cases, care should be taken on peak annotation as such low intensity peaks can also result from detector noise. However, they can be used for sequencing as long we can see at least a two to three AA sequence string, and the information obtained this way is consistent with other results. 

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