Peptide ions multiply-protonated

More recent work revealed the importance of gas phase proton transfer reactions. [91-94] This implies that multiply charged peptide ions do not exist as preformed ions in solution, but are generated by gas phase ion-ion reactions (Chap. 11.4.4). The proton exchange is driven by the difference in proton affinities (PA, Chap. 2.11) of the species encountered, e.g., a protonated solvent molecule of low PA will protonate a peptide ion with some basic sites left. Under equilibrium conditions, the process would continue until the peptide ion is saturated with protons, a state that also marks its maximum number of charges. 

Fragmentation paths yielding the c and z ions after activation of a multiply protonated peptide by ECD. 

In ECD, an ESI-produced multiply protonated peptide ion [M + H]B+ is first converted to an odd-electron [M + ion by the capture of a thermal energy electron. Subsequent transfer of H to the backbone... 

In MS sequencing, the information that describes the anuno add sequence of a peptide is contained in a product ion spectrum. This product ion spectrum is obtained in an MS-MS experiment by using CID of a protonated or multiply protonated peptide ion. Routine complete interpretation of product ion spectra to deduce the entire sequence of a peptide ion has been mainly replaced by database search programs that search large databases of spectra derived from the protein and translated gene sequences. The search identifies peptide sequences in the databases that are consistent with the spectrum. 

Hodges, B.D.M. Liang, X. McLuckey, S.A. Generation of di-hthiated peptide ions from multiply protonated peptides via ion/ion reactions. Int. J. Mass Spectrom. 2007, 267, 183-189. 

In Chapter 1 is presented a review of the instrumental requirements for the study of ion/ion reactions. Particular emphasis is given to the use of an electrodynamic ion trap for the study of multiply-protonated peptide molecules with anions. The trapped ions assume characteristic sets of m/z-dependent frequencies of motion in the oscillating quadrupole field of the ion trap, which allows ready manipulation of ions for ion isolation and activation, both of which are common elements in a tandem mass spectrometric experiment. The tandem-in-time nature of the ion trap MS" experiment provides well-defined conditions for ion/ion reactions and permits determination of ion genealogy. A bath gas, such as helium at ca 1 mTorr, intended originally to cool the ions to the center of the trap so as to enhance both sensitivity and mass resolution upon mass analysis, improves ion/ion reaction efficiencies by maximizing the spatial overlap and minimizing the translational energies of the two ion clouds. 

Electron-Capture Dissociation BCD is applicable to ESI-produced multiply charged peptide ions [101,102]. It is most conveniently implemented in an FT-ICR instrument. Multiply protonated peptides [M - - nH]"+, capture a low-energy (<0.2 eV) electron to produce an odd-electron ion [M - -nH] " +, which dissociates rapidly via an energetic H transfer to the backbone carbonyl group to form c and z sequence-specific ions ... 

Electron-transfer dissociation (ETD) is a variation of ECD in which an ion-ion reaction is conducted in a quadrnpole linear ion trap to transfer an electron to the multiply protonated peptide cation [66]. For example, anthracene anions that are generated in a Cl source have been nsed as electton donors. Analogous to the ECD process, the transfer of an electron induces fragmentation in the peptide backbone to form c- and z -type sequence-specific ions that usually retain the... 

Thus, ETD lends the capabilities of ECD to linear ion trap mass spectrometers. The individual steps involved in the operation of an LTQ instrument in ETD mode (Fig. 9.39) are injection of multiply protonated peptides as delivered by an ESI source application of a DC offset to store these ions in the front section of the LIT followed by injection of reagent anions from the Cl source into the center of the LIT. Then all but the peptide precursor ions and the electron-donor reagent ions are ejecteeL Next the DC potential well is switched off and a secondary RF voltage is applied to the end lens plates of the LIT causing positive and negative ion populations to mix and react. The reaction period is ended by axial ejection of reagent anions while positive product ions are retained in the center section of the LIT. Finally, mass-selective radial ejection as usual yields the ETD spectrum [160]. The attractive ETD technique has also been implemented on LITs with axial ejection [144,166] and on LIT-orbitrap hybrids [167-169]. 

Both ECD and ETD cause direct backbone cleavages in multiply positive peptide ions to deliver c- and z-type fragments which are highly informative for mass spectral sequencing. Especially when occurring more than once on a molecule, post-translational modifications like phosphorylation or sulfonation, reduce its tendency to form multiply protonated ions. Such analytes are best studied as negative ions, which are of course not amenable to ECD [170]. If the primary electrons... 

Zhang, X. Cassady, C. J. Apparent gas-phase acidities of multiply charged protonated peptide ions Ubiquitin, insulin B, and renin substrate. J. Am. Soc. Mass Spectrom. 1996, 7, 1211-1218. 

Gross, D. S. Williams, E. R. Experimental measurement of Coulomb energy and intrinsic dielectric polarizability of a multiply protonated peptide ion using electrospray ionization Fourier-transform mass spectrometry. J. Am. Chem. Soc. 1995, 117, 883-890. 

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 ... 

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