IR Spectroscopy of b₄ Fragment Ions of Protonated Pentapeptides in the X–H (X = C, N, O) Region

The structure of peptide fragments was studied using “action” IR spectroscopy. We report on room temperature IR spectra of b₄ fragments of protonated GGGGG, AAAAA, and YGGFL in the X–H (X = C, N, O) stretching region. Experiments were performed with a tandem mass spectrometer combined with a table top tunable laser, and the multiple photon absorption process was assisted using an auxiliary high-power CO₂ laser. These experiments provided well-resolved spectra with relatively narrow peaks in the X–H (X = C, N, O) stretching region for the b₄ fragments of protonated GGGGG, AAAAA, and YGGFL. The 3200–3700 cm^–1 range of the first two of these spectra are rather similar, and the corresponding peaks can be assigned on the basis of the classical b ion structure that has a linear backbone terminated by the oxazolone ring at the C-terminus and ionizing proton residing on the oxazolone ring nitrogen. The spectrum of the b₄ of YGGFL, on the other hand, is different from the two others and is characterized by a band observed near 3238 cm^–1. Similar band positions have recently been reported for one of the four isomers of the b₄ of YGGFL studied using double resonance IR/UV technique. As proposed in this study, the IR spectrum of this ion at room temperature can also be assigned to a linear N-terminal amine protonated oxazolone structure. However, an alternative assignment could be proposed because our room temperature IR spectrum of the b₄ of YGGFL nicely matches with the predicted IR absorption spectrum of a macrocyclic structure. Because not all experimental IR features are unambiguously assigned on the basis of the available literature structures, further theoretical studies will be required to fully exploit the benefits offered by IR spectroscopy in the X–H (X = C, N, O) stretching region

Structure of [M + H − H2O]+ from Protonated Tetraglycine Revealed by Tandem Mass Spectrometry and IRMPD Spectroscopy

Multiple-stage tandem mass spectrometry and collision-induced dissociation were used to investigate loss of H2O or CH3OH from protonated versions of GGGX (where X = G, A, and V), GGGGG, and the methyl esters of these peptides. In addition, wavelength-selective infrared multiple photon dissociation was used to characterize the [M + H − H2O]+ product derived from protonated GGGG and the major MS3 fragment, [M + H − H2O − 29]+ of this peak. Consistent with the earlier work [ Ballard, K. D. ; Gaskell, S. J. J. Am. Soc. Mass Spectrom. 1993, 4, 477 − 481 ; Reid, G. E. ; Simpson, R. J. ; O’Hair, R. A. J. Int. J. Mass Spectrom. 1999, 190/191, 209 −230 ], CID experiments show that [M + H − H2O]+ is the dominant peak generated from both protonated GGGG and protonated GGGG−OMe. This strongly suggests that the loss of the H2O molecule occurs from a position other than the C-terminal free acid and that the product does not correspond to formation of the b4 ion. Subsequent CID of [M + H − H2O]+ supports this proposal by resulting in a major product that is 29 mass units less than the precursor ion. This is consistent with loss of HN═CH2 rather than loss of carbon monoxide (28 mass units), which is characteristic of oxazolone-type bn ions. Comparison between experimental and theoretical infrared spectra for a group of possible structures confirms that the [M + H − H2O]+ peak is not a substituted oxazolone but instead suggests formation of an ion that features a five-membered ring along the peptide backbone, close to the amino terminus. Additionally, transition structure calculations and comparison of theoretical and experimental spectra of the [M + H − H2O − 29]+ peak also support this proposal