C-terminal fragment ions

As we cannot obtain any further information from the N-terminal fragments, we should try to supplement our sequence coverage with the information obtained from C-terminal fragments. First the N-terminal amino acid can be identified through a mass difference between the mass of the precursor ion and the heaviest of the y-ion... 

Fig. 1.45 Example of a 2D nano LC-MS/MS analysis of a C elegans extract. (A) Fraction 2, 4 mM KCI salt elution on the strong cation exchange column. (B) Full scan MS spectrum of the peak eluting at RT 26.3 min in (A). (C) product ion spectrum of the doubly charged precursor of (B) at m/z 784.8. Y fragments are typical for C-terminal fragments while b ions are typical for N-terminal fragments.

In the early LC-MS-MS studies, CID is performed with low-energy collisions by means of triple-quadrapole instruments. Under these conditions, series of N-terminal b-ions and C-terminal y-ions result from cleavages at the peptide bond and charge retention on either side, although double-charge tryptic peptide ions tend to favour fragmentation towards more abimdant y-ions. From these ladders of sequence ions in the MS-MS spectrum, the amino-acid sequence of the peptide may be derived. This is the bottom-up protein identification approach (Ch. 18.3.1). 

This MS-MS approach was found to be helpful in solving ambiguities in peptide identification by means of further fragmentation of especially C-terminal fragments [142], Correlation of fragment iirformation from ion-trap MS-MS and MS-MS can assist in the de novo interpretation of the MS-MS spectra [143],... 

A unique feature of ECD is that the N-terminal fragment ions, the c ions, contain an extra hydrogen atom from the proton neutralized by the electron capture. The complementarity of the c/z pair can thus be confirmed by the fact that their mass sum is 1 u greater than the Mt of the protein. 

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. 

Feng WY, Gronert S, Fletcher KA, Warres A, Lebrilla CB. The mechanism of C-terminal fragments in alkali metal ion complexes of peptides. Int J Mass Spectrom. 2003 222 117-34. 

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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