Cold, Gas-phase Uv And Ir Spectroscopy Of Protonated Leucine Enkephalin And Its Analogues

Abstract

The conformational preferences of peptide backbones and the resulting hydrogen bonding patterns provide critical biochemical information regarding the structure-function relationship of peptides and proteins. The spectroscopic study of cryogenically-cooled peptide ions in a mass spectrometer probes these H-bonding arrangements and provides information regarding the influence of a charge site. Leucine enkephalin, a biologically active endogenous opiod peptide, has been extensively studied as a model peptide in mass spectrometry. This talk will present a study of the UV and IR spectroscopy of protonated leucine enkephalin [YGGFL+H]$^{+}$ and two of its analogues: the sodiated [YGGFL+Na]$^{+}$ and C-terminally methyl esterified [YGGFL-OMe+H]$^{+}$ forms. All experiments were performed in a recently completed multi-stage mass spectrometer outfitted with a cryocooled ion trap. Ions are generated via nano-electrospray ionization and the analyte of interest is isolated in a linear ion trap. The analyte ions are trapped in a 22-pole ion trap held at 5 K by a closed cycle helium cryostat and interrogated via UV and IR lasers. Photofragments are trapped and isolated in a second LIT and mass analyzed. Double-resonance UV and IR methods were used to assign the conformation of [YGGFL+H]$^{+}$, using the NH/OH stretch, Amide I, and Amide II regions of the infrared spectrum. The assigned structure contains a single backbone conformation at vibrational/rotational temperatures of ~10 K held together with multiple H-bonds that self-solvate the NH$_{3}$$^{+}$ site. A “proton wire” between the N and C termini reinforces the H-bonding activity of the COO-H group to the F-L peptide bond, whose cleavage results in formation of the b$_{4}$ ion, which is a prevalent, low-energy fragmentation pathway for [YGGFL+H]$^{+}$. The reinforced H-bonding network in conjunction with the mobile proton theory may help explain the prevalence of the b$_{4}$ pathway. In order to elucidate structural changes caused by modifying this H-bonding activity, structural analogues were investigated. Determining the [YGGFL+Na]$^{+}$ structure will lend insight as to the impact of the ammonium group and methyl esterification of the C-terminus eliminates the carboxy proton. The talk will also report on high resolution, cold UV spectra, non-conformation specific IR gain spectra and conformation specific IR dip spectra for the analogues.