Diagnosing the Protonation Site of b₂ Peptide Fragment Ions using IRMPD in the X–H (X = O, N, and C) Stretching Region

Abstract

Charge-directed fragmentation has been shown to be the prevalent dissociation step for protonated peptides under the low-energy activation (eV) regime. Thus, the determination of the ion structure and, in particular, the characterization of the protonation site(s) of peptides and their fragments is a key approach to substantiate and refine peptide fragmentation mechanisms. Here we report on the characterization of the protonation site of oxazolone b 2 ions formed in collision-induced dissociation (CID) of the doubly protonated tryptic model-peptide YIGSR. In support of earlier work, here we provide complementary IR spectra in the 2800-3800 cm -1 range acquired on a table-top laser system. Combining this tunable laser with a high power CO 2 laser to improve spectroscopic sensitivity, well resolved bands are observed, with an excellent correspondence to the IR absorption bands of the ring-protonated oxazolone isomer as predicted by quantum chemical calculations. In particular, it is shown that a band at 3445 cm -1 , corresponding to the asymmetric N-H stretch of the (nonprotonated) N-terminal NH 2 group, is a distinct vibrational signature of the ring-protonated oxazolone structure.

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

“…Although diketopiperazines are assumed to be the thermodynamically more stable isomers, oxazolones were found to prevail in many investigations [11,15,31,32,36,38,41,42,67]. The presence of histidine can apparently lower the high barrier to formation [66] of the two cis-amide bonds in diketopiperazines [39].…”

“…The spectra in Figure 2 immediately suggest oxazolone structures for the b 2 ion of YGGFL. As the carbonyl groups have been identified as the most stable protonation sites for diketopiperazines without basic amino acid residues [15,38,41,55] Assignment labels for the theoretical spectra are inserted for vibrations referred to in the text. The conformational structures from the calculations and relative energies/kJ mol -1 (including harmonic zero point energy correction) are shown below the figure.…”

“…Room temperature IRMPD spectra in the XH stretching region were recently used to diagnose the protonation site in b 2 oxazolones [41,42], and structures with protonation at the oxazolone ring nitrogen have been reported as the major species observed in these investigations for the YI and GG b 2 ions, respectively. In contrast, under our conditions, we can largely exclude these in favor of species protonated on the N-terminal nitrogen atom for the YG b 2 ion.…”

“…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.…”

Rearrangement Pathways of the a4 Ion of Protonated YGGFL Characterized by IR Spectroscopy and Modeling

“…The majority of small b n ions are believed to have a linear backbone terminated by a five-membered oxazolone ring [5][6][7][8][9][10][11][12][13][14] at the Cterminus. Exceptions exist if amino acid side chains are involved in the formation of a variety of C-terminal ring structures [15]; typical examples include lysine [16], histidine [17][18][19], aspartic acid [20], and arginine [21] containing fragments.…”