Fig. 1: Measurement of protein turnover in vivo.

a, DELTA measures protein lifetime by sequential HTL dye capture using a HT-modified protein. (i) Before the injection of the first dye ligand (pulse), all proteins are unlabeled (gray line in graph). (ii) After injection of the pulse dye ligand (dashed green line), all proteins are labeled with the pulse dye ligand (solid green line, pulse). (iii) During the pulse–chase interval, some proteins degrade, and others are synthesized but are unlabeled. (iv) Injection of a spectrally separate chase dye ligand (dashed magenta line) binds the newly synthesized protein (solid magenta line, chase). The gray shaded area indicates where excess dye ligand delays the onset of turnover measurement, leading to a pulse overestimation error (Supplementary Text). b, The estimated lifetime error (color) as a function of dye-protein ratio (y axis) and true protein lifetime (x axis). Undersaturation (<1 dye ligand–protein ratio) causes worse errors than dye ligand excess and longer-lived proteins are estimated more accurately than short-lived ones. c,d, Turnover measurement of the nuclear protein MeCP2–HT in a knock-in (KI) mouse model. c, Experimental design: Three HTL dyes were used to measure multiple protein turnover intervals. After perfusion and dissection, coronal sections were labeled with DAPI and fluorescent antibodies to identify different cell types. d, Example field-of-view images showing the JF dyes with NeuN and DAPI for identification of neuronal nuclei. After segmentation of NeuN-positive nuclei, segmented nuclei were colored by lifetime using the sum of the two in vivo injections as the pulse (fraction pulse = (JF₆₆₉ + JF₅₅₂)/(JF₆₆₉ + JF₅₅₂ + JF₆₀₈)). e, Example coronal sections from two animals. Left and middle: images show the consistency of the lifetime estimates for aligned anteroposterior sections. Right: image shows the longer lifetime in the cerebellum (compared to middle and left images). f, MeCP2–HT neuronal nuclei lifetime (bootstrap of means from five animals and three intervals where the line is the median, boxes denote the 25th–75th percentiles and whiskers mark the 0.5th–99.5th percentiles) across CCF-aligned brain regions. a.u., arbitrary units.