Determination of picomolar dissolved free amino acids along a South Atlantic transect using reversed-phase high-performance liquid chromatography

Abstract

Dissolved free amino acids (DFAA) in seawater are a form of nitrogen (N) available for marine microbes. In oligotrophic environments where N-containing nutrients are the limiting factor for microbial growth, N nutrition from DFAA could be crucial, but as yet it is poorly resolved. Measurements of individual DFAA are challenging as concentrations are typically in the low nmol L-1 range. Here we report modifications to methodology using o-phthaldialdehyde (OPA) derivatization and reversed phase high performance liquid chromatography (HPLC) that provide a 30-fold improvement in sensitivity enabling the detection of 15 amino acids in seawater with a limit of detection as low as 10 pmol L-1 with accuracy and precision of better than 10 %. This analytical methodology is now suitable for the challenging quantitation of DFAA in oligotrophic seawaters. The method was successfully applied to a suite of seawater samples collected on a cruise crossing the South Atlantic Ocean, where concentrations of DFAAs were generally low (sub nmol L-1), revealing basin-scale features in the oceanographic distributions of DFAA. This unique dataset implies that DFAAs are an important component of the N cycle in both near-coastal and open oceans. Further calculations suggest that the proportions of organic N originating from DFAA sources were significant, contributing between 0.2-200 % that of NH 4 + and up to 77 % that of total inorganic nitrogen in the upper 400 m in some regions of the transect.

Quantification of dissolved metabolites in environmental samples through cation‐exchange solid‐phase extraction paired with liquid chromatography–mass spectrometry

“…The concentrations of many of the compounds measured in environmental samples by CX‐SPE analysis are within or close to the environmental concentration ranges reported by other studies of dissolved metabolites such as amino acids (Lee and Bada 1975; Fuhrman and Ferguson 1986; Sabadel et al 2017), TMAO (Gibb and Hatton 2004), creatine (Wawrik et al 2017), DMSP (Kiene and Slezak 2006), and arsenobetaine (Glabonjat et al 2018) (Table 1). Furthermore, the concentrations of some compounds measured for the first time using CX‐SPE such as glycine betaine were of roughly the same order of magnitude (low nM) as those predicted by uptake kinetics studies (Kiene and Slezak 2006; Boysen et al 2022; Mausz et al 2022).…”

“…CX‐SPE LODs were lower than the derivatization/GC–MS method (60–1000 nM) (Sogin et al 2019). CX‐SPE did not achieve comparable LODs for amino acids (0.025–6.8 nM) compared with focused dissolved free amino acid (DFAA) methods (0.009–0.163 nM) (Sabadel et al 2017), but CX‐SPE did still enable the quantification of many DFAAs in environmental samples. The CX‐SPE LODs for DFAAs and other compounds can potentially be improved in the future through the use of larger sample volumes and micro or nanoflow HPLC systems.…”

“…Compounds involved in microbial adaptation to salt or other stressors (e.g., compatibles solutes such as glycine betaine or DMSP) that enable organisms to handle osmotic stress and have been documented to have high concentrations in marine phytoplankton and bacteria (Welsh 2000) are also a part of our compound library. While not exhaustive, the compound library employed in this study includes many of the most abundant compounds documented in marine particulate community metabolomes (Johnson et al 2020; Boysen et al 2021; Heal et al 2021) as are compounds that are suggested by previous work to be actively cycled and potentially abundant in marine dissolved metabolomes (Gibb and Hatton 2004; Kiene and Slezak 2006; Poretsky et al 2010; Fiore et al 2015; Sabadel et al 2017; Mayali and Weber 2018; Vorobev et al 2018; Weber et al 2020; Ferrer‐González et al 2021; Widner et al 2021; Boysen et al 2022). Our in‐house library consists of 179 standards separated and analyzed using HILIC chromatography (HILIC Standards) and 70 standards separated and analyzed using RP chromatography (RP Standards) (Supporting Information Table S1).…”

Distribution Characteristics of Dissolved Amino Acids in Seawater of the Yap Trench

“…There were no significant correlations between concentrations of chl a and any amino acids (DFAA, DCAA and THAA) in this study. This result was inconsistent with other research [5,50,51], indicating that impact of plankton biomass on amino acids was very limited in the euphotic layer of the Yap Trench [35]. Amino acids and chl a are labile components in seawater with high turnover rates [44,52], and their concentrations are affected by various processes, such as phytoplankton production, biodegradation and ocean current [3,35].…”

Particulate and dissolved metabolite distributions along a latitudinal transect of the western Atlantic Ocean

“…When measured as dissolved free amino acids, phenylalanine and tryptophan display a variety of concentrations ranging from hundreds to thousands of picomolar (Mopper and Lindroth 1982) from 0 to 170 m in the Baltic Sea. A study in the south Atlantic determined that dissolved phenylalanine concentrations were in the pmol L À1 range at the southern edge of the South Atlantic Gyre but increased to low nmol L À1 concentrations closer to the coast (Sabadel et al 2017). In our study, concentrations of tryptophan and phenylalanine varied from < 10 pmol L À1 (deep ocean) to hundreds of pmol L À1 (surface ocean).…”