N-terminal fragment ions

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. 

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... 

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. 

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). 

Fragments will only be detected if they carry at least one charge. If this charge is carried by the N-terminal fragment, the ion is called either a, b, or c. If the charge... 

Additional techniques can provide further information when the structure of a peptide is unknown. One method is to carry out the protein digestion in water that is labeled with 50% 0, yielding an isotopically labeled ( 0/ 0) doublet for every cleaved peptide. When such peptides are fragmented the y ions (and other C-terminal ions) can be readily identified, as each will retain the doublet, whereas the N-terminal fragments will have normal isotope distributions that contain only the isotope. 

The X-ray structure of spinach Rubisco identifies 10 loop regions in the active-site (8). The major photolabeled peptide isolated by ion-exclusion and ion-exchange chromatography, and reverse phase HPLC corresponded to the Rubisco LSu tryptic peptide Val-42 to Arg-79 (Table 1), an active-site peptide (9). Two portions of the tryptic peptide Asp-95 to Lys-128, the peptide which comprises loop B in the crystal structure (8) were also recovered. Also recovered were two peptides (Table 1) which include residues in two other loop regions of the Rubisco active-site (8). The peptide Asp-202 to Arg-213 (loop 2 in ref. 8) is adjacent to the activator lysine (10). The peptide Gln-259 to His-267 is the N-terminal fragment of Gln-259 to Arg-285 the tryptic peptide which encompasses loop 4. Thus, all four of the photoaffinity labeled LSu peptides have been previously shown to be active-site peptides based on X-ray crystallography or chemical modification or by both techniques. 

Example I The positive-ion electtospray tandem mass spectrum of the [M+2ir ion of the P-casein tryptic phosphopeptide FQpSEEQQQTEDELQDK was obtained on a Bruker Esquire 3000 quadrupole ion trap (Fig. 9.21) [98]. Sequential amino acid residue losses from lx)th terminal ends of the peptide are labeled according to the b- and y-ion nomenclature. Due to the phosphoserine, the C- and N-terminal peptide ions (bs- bis and y - y ) exhibit an increase of 80 u in mass as compared to the unphosphorylated form. In addition, most ions are accompanied by the corresponding fragments from H3PO4 loss (98 u). 

Walters, F.S., S.L. Slatin, C.A. Kulesze, and L.H. English. 1993. Ion channel activity of n-terminal fragments from the CrylA(c) delta-endotoxin. Biochem. Biophys. Res. Comm. 196 921-926. 

The second example contained two basic amino acids in the middle of the sequence, but closer to the C-terminus. The fragmentation spectrum contained a huge number of both C- and N-terminal peaks, with a high number of doubly charged ions, including both basic amino acids. The presence of basic residues caused a more balanced number of b- and y-ions but also made it very difficult to obtain fragments with bond cleavage near basic residues. 

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