Amide I absorption spectra report on backbone structure. Here we computed the pump slice amplitude (PSA) from the 2D IR spectra at 150 fs to extract features comparable to FTIR spectra. In brief, PSA analysis projects a 1D spectrum at each excitation frequency and computes the difference between the maxima and the minima of a series of pump-probe slices . We selected a waiting time of 150 fs to avoid pulse-overlap artifacts while ensuring that peak shapes remained unaffected by energy dissipation, which occurs at longer waiting times. The PSAs of both Gal3C and Gal3C-PEG 550 Da exhibit a maximum around 1630 cm -1 (Figure ). Relative to Gal3C and Gal3C-PEG 550 Da, the maximum of Gal3C-PEG 5 kDa shows a 5 cm -1 redshift, likely indicating an increase in antiparallel β-sheet structures or changes in the backbone dihedral angles that lower the vibrational frequency. Both Gal3C-PEG 550 Da and Gal3C-PEG 5 kDa exhibit broader lineshapes from 1650 to 1680 cm -1 compared to Gal3C, but this is more Conjugation with longer PEG alters the Gal3C surface solvation network.