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.