Surveying the sequence diversity of model prebiotic peptides by mass spectrometry Jay G. Forsythea,1, Anton S. Petrova, W. Calvin Millarb,2, Sheng-Sheng Yuc, Ramanarayanan Krishnamurthyd, Martha A. Groverc, Nicholas V. Huda, and Facundo M. Fernándeza,3 aSchool of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400; bSchool of Physics, Georgia Institute of Technology, Atlanta, GA 30332-0430; cSchool of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0100; and dDepartment of Chemistry, The Scripps Research Institute, La Jolla, CA 92037 Edited by Ken A. Dill, Stony Brook University, Stony Brook, NY, and approved August 7, 2017 (received for review June 29, 2017) The rise of peptides with secondary structures and functions would (Fig. 1A). Subjecting these mixtures to sequential hot-dry/cool-wet have been a key step in the chemical evolution which led to life. As water evaporation and rehydration cycles led to depsipeptides— with modern biology, amino acid sequence would have been a peptide-like oligomers containing both amide and ester backbone primary determinant of peptide structure and activity in an origins- linkages (Fig. 1B). Ester linkages are kinetically and thermody- of-life scenario. It is a commonly held hypothesis that unique namically favored but susceptible to hydrolysis, whereas amide functional sequences would have emerged from a diverse soup of linkages are more stable; therefore, depsipeptide sequences became proto-peptides, yet there is a lack of experimental data in support of progressively enriched with amide bonds over the course of various this. Whereas the majority of studies in the field focus on peptides dry–wet cycling programs. In the absence of hydroxy acids, peptide containing only one or two types of amino acids, here we used bond formation did not spontaneously occur, confirming the plau- modern mass spectrometry (MS)-based techniques to separate and sible role of hydroxy acids as cobuilding blocks of proto-peptides. At sequence de novo proto-peptides containing broader combinations drying temperatures of 55–65 °C, depsipeptide chains contained of prebiotically plausible monomers. Using a dry–wet environmental predominantly ester bonds, whereas amide bonds became more cycling protocol, hundreds of proto-peptide sequences were formed prevalent at drying temperatures above 65 °C. Most reactions tested over a mere 4 d of reaction. Sequence homology diagrams were had an initial pH of ∼3 due to the hydroxy acid monomers present, constructed to compare experimental and theoretical sequence but depsipeptide formation also occurred at pH values of 5, 7, and 9. spaces of tetrameric proto-peptides. MS-based analyses such as this Successful depsipeptide formation with various amino acid and hy- will be increasingly necessary as origins-of-life researchers move to- droxy acid monomers was achieved, including glycine, glycolic ward systems-level investigations of prebiotic chemistry. acid, L-alanine, D-alanine, L-lactic acid, L-leucine, and L-serine. Motivated by our discovery that ester–amide exchange reactions prebiotic chemistry | chemical evolution | peptides | mass spectrometry | can produce mixed depsipeptides capable of chemical evolution, depsipeptides we sought to explore the sequence diversity of these species when more plausible mixtures of amino acid and hydroxy acid monomers n 1953, Miller demonstrated the abiotic synthesis of amino were subjected to repeated cycles. As with traditional proteomics Iacids in the now-famous spark-discharge experiment (1). Soon after, Fox and Harada began to explore the formation of peptides Significance from amino acids via condensation at high temperatures (2). These reactions were facilitated by proportionally higher concentrations Peptides and proteins are essential for life as we know it, and of amino acids with acidic side chains (e.g., aspartic acid, D) and likely played a critical role in the origins of life as well. In recent resulted in the production of “proteinoids,” condensation products years, much progress has been made in understanding plausi- containing covalent cross-links not found in coded proteins. In ble routes from amino acids to peptides. However, little is their 1960 manuscript, Fox and Harada speculated about the di- known about the diversity of sequences that could have been versity of proteinoid sequences, yet conceded that “a complete produced by abiotic condensation reactions on the prebiotic answer to the question of whether the amino acid residues are earth. In this study, multidimensional separations were cou- distributed in a random or other arrangement may require a pled with mass spectrometry to detect and sequence mixtures complete assignment of residues in one molecular species,” atask of model proto-peptides. It was observed that, starting with a beyond the analytical capabilities of the time (3). few monomers, proto-peptide diversity increased rapidly fol- In subsequent years, the proteinoid concept was displaced by lowing cycling. Experimental proto-peptide sequences were the hypothesis that either RNA or proto-RNA gave rise to life compared with theoretically random sequences, revealing a (4–6). Nevertheless, to this day, condensation reactions of amino high sequence diversity of plausible monomer combinations. acids are thought to have played a key role in a potentially symbiotic proto-nucleic acid and proto-peptide world (7, 8). Author contributions: J.G.F., A.S.P., R.K., M.A.G., N.V.H., and F.M.F. designed research; Peptide condensation studies at temperatures lower than those J.G.F., A.S.P., W.C.M., and S.-S.Y. performed research; J.G.F., R.K., M.A.G., N.V.H., and of Fox and Harada, or with the aid of chemical agents, confirmed F.M.F. analyzed data; and J.G.F. and F.M.F. wrote the paper. that abiotic production of peptides was indeed possible, but The authors declare no conflict of interest. chain lengths were generally limited to dimers and trimers with This article is a PNAS Direct Submission. low yields (9). In recent studies, this length barrier has been Data deposition: All depsipeptide sequences reported in this paper can be accessed in surpassed, and certain proto-peptides have been shown to form Dataset S1. aggregate structures (10, 11). Nevertheless, the majority of 1Present address: Department of Chemistry and Biochemistry, College of Charleston, proto-peptide studies have been limited in scope, typically con- Charleston, SC 29424. 2Present address: Master’s Industrial Internship Program, University of Oregon, Eugene, taining only one or two types of amino acid monomer. OR 97403. We recently introduced a model prebiotic pathway for peptide 3 – To whom correspondence should be addressed. Email: facundo.fernandez@chemistry. formation based on ester amide exchange reactions between gatech.edu. α α -amino acids and -hydroxy acids (12), amino acid structural This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. analogs found in meteorites and model prebiotic reactions (13, 14) 1073/pnas.1711631114/-/DCSupplemental. E7652–E7659 | PNAS | Published online August 28, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1711631114 Downloaded by guest on September 28, 2021 A C PNAS PLUS DE B R R R generic depsipeptide CHEMISTRY Fig. 1. Experimental overview and example dataset. (A) Hydroxy and amino acid monomers used in this study. Hydroxy acids are abbreviated with lowercase letters corresponding to their analogous amino acids (e.g., glycolic acid = g; glycine = G). All chiral species were in their l form only. (B) Depsipeptides formed by ester–amide exchange contained a mixture of ester and amide backbone linkages and were initiated by a hydroxy acid at the “N” terminus. Depsipeptides were formed from hydroxy acid and amino acid mixtures by alternating polymerizing (85 °C dry-down, 18 h, open vial) and hydrolyzing (65 °C aqueous, 6 h, closed vial) conditions. Each cycle consisted of one polymerization and one hydrolysis step over a period of 24 h. (C) General workflow of depsipeptide separation and sequencing. Depsipeptides were separated by UPLC and traveling-wave IM (TWIM) before data-dependent MS/MS acquisition. To evaluate MS/MS data, an algorithm was developed which created all possible sequences (based on monomer inputs and polymer length). Theoretical MS/MS spectra were generated and were matched to experimental data using commercial MS software. (D) Example UPLC chromatogram of a mixture of a-, G-, A-, and BIOCHEMISTRY L-containing depsipeptides formed after four dry-wet cycles. (Inset) The extracted TWIM signal of m/z 231.11, which eluted at 9.4 min, is shown. (E) Negative- mode MS/MS spectrum of m/z 231.11 from D. The resulting sequence is aAA. research, mass spectrometry (MS) was the logical choice for the mental data were matched with theoretical sequences containing investigation of depsipeptide primary structure (i.e., monomer se- hydroxy acids and amino acids via accurate mass and tandem mass quence) in the complex reaction mixtures. Significant develop- spectral similarity searches. Using this workflow, hundreds of dep- ments in MS and ancillary technologies (15, 16) have dramatically sipeptide sequences were surveyed after only 4 d of cycling. Experi- advanced the ability to deeply characterize biological peptidomes mentally observed depsipeptide sequences were compared with the and proteomes, with MS-based methods enabling the detection of theoretically possible permutations using homology diagrams in