Kinetics of a collagen-like polypeptide fragmentation after mid-IR free-electron laser ablation

Abstract

Tissue ablation with mid-infrared irradiation tuned to collagen vibrational modes results in minimal collateral damage. The hypothesis for this effect includes selective scission of protein molecules and excitation of surrounding water molecules, with the scission process currently favored. In this article, we describe the postablation infrared spectral decay kinetics in a model collagen-like peptide (Pro-Pro-Gly)(10). We find that the decay is exponential with different decay times for other, simpler dipeptides. Furthermore, we find that collagen-like polypeptides, such as (Pro-Pro-Gly)(10), show multiple decay times, indicating multiple scission locations and cross-linking to form longer chain molecules. In combination with data from high-resolution mass spectrometry, we interpret these products to result from the generation of reactive intermediates, such as free radicals, cyanate ions, and isocyanic acid, which can form cross-links and protein adducts. Our results lead to a more complete explanation of the reduced collateral damage resulting from infrared laser irradiation through a mechanism involving cross-linking in which collagen-like molecules form a network of cross-linked fibers.

FIGURE 1

Optical microscope images of (Pro-Pro-Gly)₁₀ target surface (area size is 50 × 50 μm). (A) Nonablated and (B) ablated at amide II (6.45 μm). Sample color changed from white transparent to yellow/brown. At higher FEL fluencies, the crystals are converted to liquid/gel.

FIGURE 2

IR absorption spectra of the collagen-like polypeptides (Pro-Pro-Gly)₁₀, (Pro-Hyp-Gly)₁₀, and their components (dipeptides and single amino acids) after low energy ablation. Vertical arrows indicate FEL wavelengths, which were used for the ablation.

FIGURE 3

Area of interest in the IR absorption spectra of studied compounds and lysozyme (for comparison), taken at 40 s after ablation.

FIGURE 4

Kinetic series of IR absorption spectra after ablation, showing decay of the 2160 cm⁻¹ peak in (Pro-Pro-Gly)₁₀, (Pro-Hyp-Gly)₁₀, and their component dipeptides.

FIGURE 5

Proposed model for formation of cyanate ions O=C=N(-) and isocyanic acid (O=C=N-H) in the FEL ablation by homolytic cleavage of a collagen-like polypeptide (Pro-Pro-Gly)₁₀ at an internal glycine residue. The resultant peptidyl radicals can initiate further free radical reactions. The O=C=N(-) and O=C=N-H intermediates exhibit volatile and nonvolatile IR peaks, correspondingly.

FIGURE 6

Total ion chromatograms: (A) (Pro-Pro-Gly)₁₀ degradation products resulting from FEL irradiation at 3.4 μm; (B) original (Pro-Pro-Gly)₁₀ peptide. These data were acquired in low mass resolution mode on an LTQ ion trap MS as described in Materials and Methods.

FIGURE 7

(A) Base peak ion chromatogram of (Pro-Pro-Gly)₁₀ degradation products resulting from irradiation at 7.17 μm. (B) Extracted ion profile of m/z 1266.15 for the cross-linked trimer (Pro-Pro-Gly)₁₀ peptide with no carbamyl adducts. (C) Extracted ion profile of m/z 1273.30 for the molecule with one carbamyl adduct. (D) Extracted ion profile of m/z 1280.65 for the molecule with two carbamyl adducts. These data represent the multiply charged precursor ions (z = 6) acquired in high resolution mode (60,000 FWHM) using the Orbitrap MS. The presence of multiple chromatographic peaks for each cross-linked species indicates multiple sites of cross-link attachment.

FIGURE 8

Signal averaged precursor spectrum (t = 42.2–45.3 min) for the various carbamyl adducts. These data were acquired in high resolution mode with the Orbitrap MS and show both the more dominant +6 charge state and the weaker +7 charge state. The mass range of the instrument was not set to identify lower charge state ions (z < 6) at higher m/z values.

FIGURE 9

(A) High resolution mass spectrum of the z = +6 ion corresponding to the N-carbamyl-(Pro-Pro-Gly)₁₀ cross-linked product [3(Pro-Pro-Gly)₁₀ + 1 carbamyl + 6H⁺]⁶⁺. (B) Theoretical isotopic distribution corresponding to the carbamyl adduct with the molecular formula (C₃₆₁ H₅₂₃ N₉₁ O₉₄) for the cross-linked adduct. The monoisotopic mass of the most abundant isotopic peak (m/z 1273.4885) was nearly identical to that calculated (m/z 1273.4837) for the predicted isotopomer spectrum to within 3.77 ppm. The observed isotopic distribution pattern is essentially identical to the theoretical spectrum. The monoisotopic mass (z = 1) of this adduct is 7631.8545.

FIGURE 10

Proposed model for formation of cross-links between collagen-like polypeptide (Pro-Pro-Gly)₁₀ molecules by free radical reactions among FEL-generated resultant peptidyl radicals.