Fig. 2

Fig. 2 TMR-O shows superior selective binding to protein and tissue aldehydes.

Created in part with Biorender.com. a Binding of the probes to aldehyde-rich protein (BSA-Ald) was determined by incubating the probes (10 mM) with BSA-Ald (25 mM protein, 100 mM aldehyde) with and without aldehyde-blocking with methoxylamine. After incubation at 37 °C for 3 h, free and protein-bound probe were separated by passing solutions through a 10 kD molecular weight cut-off (MWCO) filter. b UV-Vis spectra of a solution of TMR-O incubated with BSA-Ald before (blue) and after (red) passing through a MWCO filter show a significant decrease in the TMR-O signal due to binding to BSA-Ald. (c) Blocking of the aldehyde-reactive sites on BSA-Ald obstructs TMR-O binding, resulting in little change in the UV-Vis absorption of TMR-O after filtration. d, e Quantification of the percent binding of aldehyde-reactive TAMRA-based probes to BSA-Ald without (d) and with (e) methoxamine pre-blocking demonstrates their specificity for aldehydes. f Lysine aldehydes (Lys^Ald) are abundant in healthy mammalian aorta and zebrafish bulbus due to elastin turn-over and cross-linking (top). These tissues, therefore, provide a robust control to characterize the accuracy of TAMRA-based aldehyde-reactive probes for lysine aldehydes such as allysine and hydroxy-allysine. g, h White light and fluorescence reflectance images of whole mouse (g) and zebrafish (h) hearts excised after systemic injection of TMR-O or TMR-NB. TMR-O binds specifically to the aorta and bulbus, while the non-binding control probe TMR-NB produces no signal. Imaging was performed in 3 mice/fish per group with consistent results. i Quantification of fluorescence signal intensity in the zebrafish bulbus 4 h after IP injection of an allysine-binding probe (2 nmol/g) shows > 2-fold higher signal for TMR-O compared to other probes and no uptake of TMR-NB. Signal is normalized to the fluorescence signal of a phantom made from the injected dose of each probe. Data are means ± SD of three independent experiments. Source data are provided in the Source Data file.