Proteins doubly charged ions

The MALDI mass spectrum obtained from the peptides resulting from the enzymatic digestion (Asp-N) of the bFGF protein (A) spectrum before and (B) spectrum after immunoprecipitation with an antibody directed against the recombinant protein. The peaks marked correspond to either Cu adducts or doubly charged ions. (C) Schematic representation of the observed peptides. A continuous line corresponds to a peptide that links to the antibody, and a dotted line to a peptide that does not link. Reproduced (modified) from Zhao Y.M., Muir T.W., Kent S.B.FL, Tisher E., Scardina J.M. and Chait B.T., Proc. Natl. Acad. Sci. USA, 93, 4020M024, 199, with permission. 

FIGURE 36.16 FT-ICR-MS/MS spectrum obtained from an archaeological protein extract that illustrates the y and b fragments of the myoglobin peptide (VETDLAGHGQEVLIR) via the doubly charged ion at miz 818.936 [146]. 

Figure 35. Doubly charged ions from tryptic digests of protein.s...

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. 

The most important multiply charged polyatomic positive ions are compounds with two or more basic groups which when protonated lead to doubly or poly-charged ions. Typical examples are diamines such as the double protonated a, to alkyldiamines, H3N(CH2)pNH2+, and the most important class, the polyprotonated peptides and proteins, which have multiple basic residues. Charge reduction for these systems occurs through proton transfer from one of the protonated basic sites to a solvent molecule. Such a reaction is shown below for the monohydrate of a doubly protonated diamine ... 

The imidazole side-chain of histidine has a value of 6.0, making it a weaker base than the unsubstituted imidazole. This reflects the electron-withdrawing inductive effect of the amino group, or, more correctly the ammonium ion, since amino acids at pH values around neutrality exist as doubly charged zwitterionic forms (see Box 4.7). Using the Henderson-Hasselbalch equation, this translates to approximately 9% ionization of the heterocyclic side-chain of histidine at pH 7 (see Box 4.7). In proteins, plCa values for histidine side-chains are estimated to be in range 6-7, so that the level of ionization will, therefore, be somewhere between 9 and 50%, depending upon the protein. 

Covey, T. R. Huang, E. C. Henion, J. D. 1991. Structural characterization of protein tryptic peptides via hquid chromatography/mass spectrometry and collision-induced dissociation of their doubly charged molecular ions. Anal. Chem., 63,1193-1200. 

In general, MALDI of samples fixed to membranes resulted in no loss of mass resolution or mass range. Spectra were extremely reproducible, and could usually be acquired at a lower threshold laser intensity. Figure 1 shows a peptide and protein mixture desorbed from each of the 5 tested membranes. All membranes except the CIS extraction disk produced well resolved spectra. Higher masses were better resolved in samples fixed to polyethylene membranes, while lower masses were better resolved by fixing samples to the Type 61 disposable IR card. Doubly and triply charged ions formed more readily upon desorption/ionization from all the membranes tested, than from stainless steel surfaces. 

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