Towards Understanding the Tandem Mass Spectra of Protonated Oligopeptides. 2: The Proline Effect in Collision-Induced Dissociation of Protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp)

Abstract

The product ion spectra of proline-containing peptides are commonly dominated by y n 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 y 2 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 y 2 /b 3 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 y 2 /b 3 abundance ratio decreases.

Single-photon absorption of isolated collagen mimetic peptides and triple-helix models in the VUV-X energy range

“…Most single backbone cleavages occur N-terminal to proline residues. This preferential cleavage site (the so-called proline effect) has been observed previously in CID or UV photofragmentation of proline-containing peptides [31][32][33] . This is consistent with the fact that in our experiment, these fragments are mainly formed at low energy when photoexcitation without ionization dominates.…”

“…We can infer that the higher fragmentation yield of [(PHypG) 10 +3H] 3+ compared to [(PHypG) 10 +2H] 2+ is mainly due to the additional proton, which can be transferred at a backbone N and induce bond cleavage. This "mobile proton" mechanism is widely known in CID of protonated peptides 34 , and has been used to explain the proline effect 33,35,36 . The dominance of multiple backbone cleavages at high photon energy can be attributed to increasing internal energy transferred to the peptides after photoabsorption, leading to further dissociation of the fragments formed by single backbone bond cleavage.…”

Relative Stability of Peptide Sequence Ions Generated by Tandem Mass Spectrometry

“…A different yardstick is potentially necessary for DFT, but not easy to test, because most of the peptide systems studied with BIRD are enormous from a DFT calculation point of view. Anecdotally, BIRD provides the [YGGFL + H] + barrier as 106.7 kJ mol -1 [48], which is substantially lower than barriers determined for the fragmentation of [AAAPA + H] + by DFT (115.9 kJ mol -1 [77] (same level of theory as used here) and the lowest barrier available in the literature for such calculations). That P-containing peptide has a wellestablished low barrier residue specific bond cleavage (for comparison [AAAAA + H] + is 135.6 kJ mol -1 [71]) unlike YGGFL, but the barrier is still higher than that from BIRD.…”

Conformation-Specific Spectroscopy of Peptide Fragment Ions in a Low-Temperature Ion Trap

“…Different tools, sometimes in combination, have been applied for obtaining information on the structures of peptide fragment ions, including theoretical calculations [9,10], multi-stage mass spectrometry (MS/MS, or MS n ) [3,4,[11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28], ion mobility spectrometry (IMS) [29,30], hydrogen-deuterium exchange (HDX) [22,31,32], and infrared multiple photon dissociation (IRMPD) spectroscopy [5,22,[31][32][33][34][35][36][37][38][39][40][41][42]. While spectroscopy is a particularly powerful way to obtain detailed information about the structures of gas phase molecules, approaches based on IRMPD feature certain drawbacks.…”