Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of model prebiotic peptides: Optimization of sample preparation.

RATIONALE: Depsipeptides, or peptides with a mixture of amide and ester linkages, may have evolved into peptides on primordial Earth. Previous studies on depsipeptides utilized electrospray ionization ion mobility quadrupole time-of-flight (ESI-IM-QTOF) tandem mass spectrometry; such analysis was thorough yet time-consuming. Here, a complementary matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) approach was optimized for rapid characterization of depsipeptide length and monomer composition. METHODS: Depsipeptide mixtures of varying hydrophobicity were formed by subjecting aqueous mixtures of α-hydroxy acids and α-amino acids to evaporative cycles. Ester and amide content of depsipeptides was orthogonally confirmed using infrared spectroscopy. MALDI-TOF MS analysis was performed on a Voyager DE-STR in reflection geometry and positive ion mode. Optimization parameters included choice of matrix, sample solvent, matrix-to-analyte ratio, and ionization additives. RESULTS: It was determined that evaporated depsipeptide samples should be mixed with 2,5-dihydroxybenzoic acid (DHB) matrix in order to detect the highest number of unique signals. Low matrix-to-analyte ratios were found to generate higher quality spectra, likely due to a combination of matrix suppression and improved co-crystallization. Using this optimized protocol, a new depsipeptide mixture was characterized. CONCLUSIONS: Understanding the diversity and chemical evolution of proto-peptides is of interest to origins-of-life research. Here, we have demonstrated MALDI-TOF MS can be used to rapidly screen the length and monomer composition of model prebiotic peptides containing a mixture of ester and amide backbone linkages.

Synthesis of ß-Peptide Standards for Use in Model Prebiotic Reactions.

A one-pot method was developed for the preparation of a series of ß-alanine standards of moderate size (2 to ≥12 residues) for studies concerning the prebiotic origins of peptides. The one-pot synthesis involved two sequential reactions: (1) dry-down self-condensation of ß-alanine methyl ester, yielding ß-alanine peptide methyl ester oligomers, and (2) subsequent hydrolysis of ß-alanine peptide methyl ester oligomers, producing a series of ß-alanine peptide standards. These standards were then spiked into a model prebiotic product mixture to confirm by HPLC the formation of ß-alanine peptides under plausible reaction conditions. The simplicity of this approach suggests it can be used to prepare a variety of ß-peptide standards for investigating differences between α- and ß-peptides in the context of prebiotic chemistry.