Immunoanalytical Approach for Detecting and Identifying Ancestral Peptide Biomarkers in Early Earth Analogue Environments Severino, Rita Risso, Valeria Alejandra Sánchez Ruiz, José Manuel The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.2c05386. Detailed description of experimental procedures. Illus- tration of antibodies characterization, other FSMI results, inhibition immunoassays, target epitopes, samples, and metagenomics and metaproteomics results. Tables with sample descriptions, antibody character- ization (limits of detection and coefficient of variance), and statistics results (PDF) https://pubs.acs.org/doi/suppl/10.1021/acs.analchem.2c05386/suppl_file/ac2c05386_si_001.pdf We thank Dr. Eric Gaucher (Georgia State University, U.S.A.) and Dr. Miguel Alcalde (Instituto de Catálisis y Petroleoquí- mica, CSIC, Spain) for providing ancestral Elongation Factor Tu and RuBisCO proteins, respectively. We also thank Dr. Laura Barrios, Responsible for the Statistics Unit at the Spanish National Research Council, Madrid (CSIC), for her invaluable help with statistics. We also thank the Complejo Turístico Tatio Mallku (Chile) for allowing access and sampling in the El Tatio geysers field. This research has been funded by Grant Nos. RTI2018-094368-B-I00 and PID2021-126746NB-I00 by the Spanish Ministry of Science and Innovation/State Agency of Research MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. R.S. is a Ph.D. fellow from INTA, F.P.-S. was supported by the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Grant Agreement No. 892961, and V.A.R. was supported by Junta de Andalucia-Consejería de Economía y Conocimiento (PAIDI 2020) P21_00112. The field campaign to El Tatio was funded by the CAN7 project of the NASA Astrobiology Institute (Ref 13-13NAI7_2-0018). The metaproteome sequencing and analysis was performed in the Proteomics Unit of the Universidad Complutense de Madrid, supported by Grant PT17/0019, of the PE I+D+i 2013-2016, funded by the Instituto de Salud Carlos III (ISCIII) and the European Regional Development Fund (ERDF). Several mass spectrometry and spectroscopic techniques have been used in the search for molecular biomarkers on Mars. A major constraint is their capability to detect and identify large and complex compounds such as peptides or other biopolymers. Multiplex immunoassays can detect these com-pounds, but antibodies must be produced for a large number of sequence-dependent molecular targets. Ancestral Sequence Re-construction (ASR) followed by protein "resurrection" in the lab can help to narrow the selection of targets. Herein, we propose an immunoanalytical method to identify ancient and universally conserved protein/peptide sequences as targets for identifying ancestral biomarkers in nature. We have developed, tested, and validated this approach by producing antibodies to eight previously described ancestral resurrected proteins (three beta-lactamases, three thioredoxins, one Elongation Factor Tu, and one RuBisCO, all of them theoretically dated as Precambrian), and used them as a proxy to search for any potential feature of them that could be present in current natural environments. By fluorescent sandwich microarray immunoassays (FSMI), we have detected positive immunoreactions with antibodies to the oldest beta-lactamase and thioredoxin proteins (ca. 4 Ga) in samples from a hydrothermal environment. Fine epitope mapping and inhibitory immunoassays allowed the identification of well-conserved epitope peptide sequences that resulted from ASR and were present in the sample. We corroborated these results by metagenomic sequencing and found several genes encoding analogue proteins with significant matches to the peptide epitopes identified with the antibodies. The results demonstrated that peptides inferred from ASR studies have true counterpart analogues in Nature, which validates and strengthens the well-known ASR/protein resurrection technique and our immunoanalytical approach for investigating ancient environments and metabolisms on Earth and elsewhere. 2023-05-12T12:11:07Z 2023-05-12T12:11:07Z 2023-03-16 journal article Severino, R. et al. Immunoanalytical Approach for Detecting and Identifying Ancestral Peptide Biomarkers in Early Earth Analogue Environments. Anal. Chem. 2023, 95, 5323−5330. [https://doi.org/10.1021/acs.analchem.2c05386] https://hdl.handle.net/10481/81495 10.1021/acs.analchem.2c05386 eng info:eu-repo/grantAgreement/EC/H2020/Marie Sklodowska-Curie 892961 http://creativecommons.org/licenses/by-nc/4.0/ open access Atribución-NoComercial 4.0 Internacional American Chemical Society