Coupling gel electrophoresis with photoluminescence reveals biochrome complexes in modern and fossil shells

1. Polyacrylamide gel electrophoresis is commonly used to visualize mixtures of proteins, such as those embedded in calcareous biominerals. However, it is ineffective for the detection of biochromes, a major class of low molecular weight organic compounds commonly associated with calcified exoskeletons. 2. We describe a novel approach based on the coupling of electrophoresis and photoluminescence (PL) spectral imaging to reveal invisible biochromes, identify them chemically, provide evidence for their putative interaction with exoskeletal macromolecules and purify them in large amounts. Our protocol relies on three key-steps: a mild extraction of all organics from bleached skeletal powder (step 1); an optimized electrophoretic fractionation immediately followed by direct on-gel spectral image acquisition performed before classical gel staining (step 2); a large-scale purification via preparative electrophoresis coupled to PL spectral imaging, to obtain significant amounts of biochromes of interest (step 3). 3. Steps 1–3 were successfully applied to recent gastropod shell extracts, while steps 1 and 2 were applied to their fossil equivalents from the Eocene epoch (≈approximately 45 million years). In both cases, we were able to identify porphyrins associated with the shells via a spectrophotometric measurement of the luminescent bands directly on the analytical gel, even at low concentration in the case of fossil extract. For recent shell extracts, our approach allowed us to identify spectrophotometrically the tubes containing the invisible pigments, after preparative fractionation and fraction collection. 4. Our protocol enables direct on-gel identification of porphyrin molecules, even in trace amounts. It opens up new avenues for studying a wide range of biological composites, whether mineralized or not, that contain photoluminescent biochromes. It is particularly well-suited to ancient specimens and fossils to trace the origin and evolution of biochrome complexes in the geological record.