Improving Molecular Formula Assignment of Nitrogen- and Sulfur-Rich DOM in Ultrahigh-Resolution Mass Spectrometry: Validation of Oligopeptides

Abstract

Abstract Molecular formula (MF) assignment in ultrahigh-resolution mass spectrometry (UHRMS) analyses of dissolved organic matter (DOM) containing multiple non-oxygen heteroatoms (e.g., nitrogen and sulfur) is highly environmentally relevant yet remains challenging. Recent studies of DOM composition in aquaculture systems with protein-rich surface sediments have reported numerous CHNO (N1−8), CHOS (S1−3), and even CHNOS MFs containing multiple N and S atoms, suggesting the presence of oligopeptide-like molecules. Due to insufficient mass resolution, multiple formula assignments (MultiAs) caused by interchangeable replacement pairs, within a mass error threshold of 1 ppm, complicate the validation of MF assignments. While N5S2 formulas are known as likely false-positive assignments of 13C1 isotopologue peaks, the multiple N-containing MFs in MultiAs of potential oligopeptide origins involving replacement pairs with N6 ↔ S1 and S2 ↔ N4 could not be validated properly. Here, we introduce a new chemically informed validation approach based on the elemental composition and stoichiometry of typical peptides with atomic ratios 0.2 < O/C < 0.65 and 1.5 < H/C < 2. Amino acid combinations matched about 36% N-rich MF (n = 960) among N6 ↔ S1 related MultiAs (N7 ↔ N1S1, N8 ↔ N2S1··· N8S2 ↔ N2S3) and 30% N-rich MF (n = 1040) among S2 ↔ N4 related MultiAs (N1S2 ↔ N5, N2S2 ↔ N6···N4S3 ↔ N8S1), supporting the true assignments of N-rich MFs based on oligopeptide stoichiometry. Overall, the chemical complexity of heteroatom-rich DOM warrants advances in MF assignment for UHR mass spectra by expanding the analytical window to include multiple-heteroatom formulas, which would especially benefit protein-rich samples from aquaculture systems or wastewater.