Towards Understanding the Tandem Mass Spectra of Protonated Oligopeptides. 2: The Proline Effect in Collision-Induced Dissociation of Protonated Ala-Ala-Xxx-Pro-Ala

The product ion spectra of proline-containing peptides are commonly dominated by yn ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-AlaXxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/timeof-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated AlaAla-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y2 ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y2/b3 abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y2/b3 abundance ratio decreases.