First sequencing of ancient coral skeletal proteins

Abstract

Here we report the first recovery, sequencing, and identification of fossil biomineral proteins from a Pleistocene fossil invertebrate, the stony coral Orbicella annularis. This fossil retains total hydrolysable amino acids of a roughly similar composition to extracts from modern O. annularis skeletons, with the amino acid data rich in Asx (Asp + Asn) and Glx (Glu + Gln) typical of invertebrate skeletal proteins. It also retains several proteins, including a highly acidic protein, also known from modern coral skeletal proteomes that we sequenced by LC–MS/MS over multiple trials in the best-preserved fossil coral specimen. A combination of degradation or amino acid racemization inhibition of trypsin digestion appears to limit greater recovery. Nevertheless, our workflow determines optimal samples for effective sequencing of fossil coral proteins, allowing comparison of modern and fossil invertebrate protein sequences, and will likely lead to further improvements of the methods. Sequencing of endogenous organic molecules in fossil invertebrate biominerals provides an ancient record of composition, potentially clarifying evolutionary changes and biotic responses to paleoenvironments.

Fossil Corals With Various Degrees of Preservation Can Retain Information About Biomineralization-Related Organic Material

“…For instance, we can with some confidence assign the Pleistocene Montastraea cavernosa and Orbicella annularis corals to stage 5e of the Key Largo formation (Hendy et al, 2020); however, we cannot absolutely rule out that it was actually derived from stage 4 (Multer et al, 2002;Muhs et al, 2011). Elemental, mineralogical, amino acid, and proteomic data for two fossil O. annularis specimens and one M. cavernosa have been previously reported (Drake et al, 2020b).…”

“…Fragments were cleaned of organic material in equal parts 3% bleach and 50% hydrogen peroxide, milled or ground, and cleaned three further times in bleach/hydrogen peroxide for 30 min (milled powder) or overnight (ground powder) each cleaning followed by copious rinsing using modified methods of (Stoll et al, 2001). Several checks of the final product suggest that only endogenous intracrystalline SOM remained (Drake et al, 2020b). Samples for XRD were milled using a #2 carbide bur while samples for amino acid analysis were ground to <125 um on an agate mortar and pestle and sieved.…”

“…Next we took a targeted approach to the amino acid data from all fossils, including Z. dalli, and some modern coral skeletons used in previous coral skeletal proteomics analyses (Drake et al, 2020b) using principal component analysis (PCA). We examined the amino acid relative composition (% THAA and FAA Asx, Glx), fraction of THAA that is FAA, total pmol amino acids per mass of skeleton, racemization (D/L THAA and D/L FAA Asx, Glx, and Ser) and THAA and FAA L-Ser/L-Asx across the samples.…”

“…Of these matrix components, proteins are the best studied; their preservation in the mineral can lead to inferences of function and provide indirect genetic information in the absence of preserved DNA (e.g., Schroeter et al, 2017). Recent analyses have shown that intact endogenous peptides remain in invertebrate skeletons for 1000s-100,000s of years (Sakalauskaite et al, 2019;Drake L. et al, 2020b;Sakalauskaite et al, 2020). Across various taxa, intact peptides may retain amino acids longer in fossil specimens than if those biomolecules exist individually as free amino acids (Bada et al, 1999), allowing for detection of ecological and ocean chemistry signatures including the occurrence of photosymbiosis in fossil scleractinian corals (Muscatine et al, 2005;Tornabene et al, 2017) and past local nitrogen cycling regimes (Wang et al, 2015).…”

“…Nevertheless, the utility of either component of a fossil coral, inorganic or organic, is limited by its preservation (Nothdurft et al, 2007;Rabier et al, 2008). Fossil corals can undergo submarine and/or subaerial post-depositional alterations including dissolution of primary aragonite, precipitation of secondary aragonite cements and/or high-Mg calcite, and recrystallization from aragonite to calcite, with subsequent alteration of the original aragonitic geochemical signature (Brand and Veizer, 1980;Sayani et al, 2011;Griffiths et al, 2013) and potential degradation of proteins with loss of amino acids (Tomiak et al, 2013;Tomiak et al, 2016;Drake et al, 2020b). All primary aragonite corals with minimal macro-and micro-structural inorganic degradation are considered equally, but partially recrystallized corals are often wholly excluded from bulk analysis (e.g., Gothmann et al, 2015), or their recrystallized portions are removed prior to further analysis (e.g., Tornabene et al).…”