Leucine-enkephaline, mass spectrum

Comparison of e contour plot from the chymotryptic digest of phosphorylase to that after re-acetylation revealed only two ions shared by both (Fig. 3). One peptide eluted at about 20 minutes with an m/z of 556.6, again representing the leucine enkephalin internal standard. The other shared ion eluted at about 12 minutes with m/z 514.5. Tandem mass spectrometry of this ion gave a CID spectrum (Fig. 4) consistent with the amino terminal four amino acids, including the acetylated (natural abundance) amino terminal serine. This indicates that the chymot sin cleaved at the fourtii residue, a leucine. 

FIGURE 47.5 Representative mass spectrum of leucine-enkephalin obtained using chip with open channel emitter (a) and porous polymer monolith-assisted electrospray process from a microdevice at a constant infusion (b). (Reprinted with permission from Koerner, T. and Oleschuk, R. D Rapid Comm. Mass Spectr., 19, 3279, 2005. Copyright 2005 J. Wiley.) Conditions peptide solution 1.0 p-mol/L in 50% aqueous acetonitrile with 0.5% acetic acid, flow rate of 100 nL/min, applied voltage of 3.5 and 3.0 kV, respectively. 

Figure 8. Partial hard ionization mass spectrum of leucine-enkephaline.

As indicated in the mass spectrum of leucine-enkephaline the most intense signal, leading to the base peak, is due to a side chain reaction... 

In order to simplify further the identification of the N-terminus-containing ions. Hurt and Morris have suggested the use of stable isotopes [29]. If the N-acetylation of leucine-enkephalin is performed with a 1 1 mixture of and acetic anhydride in methanol, the mass spectrum of the product consists of doublets... 

Figure 3. The FAB mass spectrum of leucine-enkephalin in glycerol.

Figure 5. The FAB mass spectrum of 1 1 H3/D3 N-acetyl leucine-enkephalin in glycerol -F 1 /il of oxalic acid-saturated water.

Figure 7 shows the spectrum obtained from the methyl ester of leucine-enkephalin. The ester was prepared by dissolving approximately 300 yg of dry peptide directly in 150 y of a fresh solution of HCl-saturated methanol (pH a 1). The reaction vial was sealed and heated at 42 °C for 3h, and the mass spectrum was obtained from I /A of the reaction mixture (a 2yg of the derivative) dissolved in glycerol. 

The spectrum of the leucine-enkephalin methyl ester shows a series of diagnostic 14 dalton shifts for the C-terminus-containing fragment ions. In addition, the molecular ion has shifted positively by 14 daltons, consistent with the single reactive cabonyl group in the molecule. In contrast to the previously shown N-acetylation results, methyl esterification produces shifts in the complementary set of ions as shown below. By using N-acetyl and methyl ester derivatives one can obtain sequence information from both the N- and C-terminus of a peptide, while at the same time improving FAB mass spectral performance. 

Other less common fragmentation pathways, e.g., resulting in internal fragments or neutral loss ions, are not discussed here. As an example we can look at the neuropeptide leucine enkephalin, which has a sequence of yggfl and a protonated monoisotopic mass of mh 556.28. The fragmentation of this ion in a collision cell through CAD might produce a tandem mass spectrum similar to the one in Eig. 3. 

Figure 29.28 The mass spectrum of leucine enkephalin (C2gH3 N50 ), a peptide made up of five amino acids. It has been charged by adding a proton instead of by ionisation by high-energy electrons (which would fragment the molecule). This is known as a soft ionisation method.

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