Identification to species level of live single microalgal cells from plankton samples with matrix-free laser/desorption ionization mass spectrometry

Abstract

Introduction Marine planktonic communities are complex microbial consortia often dominated by microscopic algae. The taxonomic identification of individual phytoplankton cells usually relies on their morphology and demands expert knowledge. Recently, a live single-cell mass spectrometry (LSC-MS) pipeline was developed to generate metabolic profiles of microalgae. Objective Taxonomic identification of diverse microalgal single cells from collection strains and plankton samples based on the metabolic fingerprints analyzed with matrix-free laser desorption/ionization high-resolution mass spectrometry. Methods Matrix-free atmospheric pressure laser-desorption ionization mass spectrometry was performed to acquire singlecell mass spectra from collection strains and prior identified environmental isolates. The computational identification of microalgal species was performed by spectral pattern matching (SPM). Three similarity scores and a bootstrap-derived confidence score were evaluated in terms of their classification performance. The effects of high and low-mass resolutions on the classification success were evaluated. Results Several hundred single-cell mass spectra from nine genera and nine species of marine microalgae were obtained. SPM enabled the identification of single cells at the genus and species level with high accuracies. The receiver operating characteristic (ROC) curves indicated a good performance of the similarity measures but were outperformed by the bootstrapderived confidence scores. Conclusion This is the first study to solve taxonomic identification of microalgae based on the metabolic fingerprints of the individual cell using an SPM approach.

Chemotaxonomic patterns in intracellular metabolites of marine microbial plankton

“…While some of these metabolites have reported observations in seawater, we have yet to resolve their distributions, functions, and cycling in natural microbial populations. New analytical approaches including single-cell imaging metabolomics allow resolution of metabolites exchanged in microbial consortia (Baumeister et al, 2020;Vallet et al, 2021). Analytical methods have also recently become available to measure polar metabolites like sulfonates, amino acids, and sugars in the dissolved fraction of seawater (Widner et al, 2021), opening up new avenues to compare concentrations of intracellular and excreted metabolites relevant to nutrient cycling.…”

“…Isotope-based tools like Chip-SIP have provided highthroughput means to quantify taxon-specific incorporation of stable isotope-labeled substrates (Mayali et al, 2012). Highresolution metabolomics studies of cultures and field samples have demonstrated the value of metabolic fingerprints in distinguishing plankton (Baumeister et al, 2020) and identified underexplored sulfur-containing metabolites gonyol, dimethylsulfonioacetate (DMSAc), dimethylsulfoxonium propionate (DMSOP) (Gebser and Pohnert, 2013;Thume et al, 2018;Gebser et al, 2020) and nitrogen-containing metabolites trigonelline and homarine (Heal et al, 2021) implicated in metabolic exchange among microbial taxa.…”

Integrative Description of Cryptic Tigriopus Species From Korea Using MALDI-TOF MS and DNA Barcoding

“…This identification method was initially used for the identification of species of bacteria, fungi, and viruses (e.g., Claydon et al, 1996;Fenselau and Demirev, 2001), and is still widely used (e.g., Seng et al, 2010;Chalupová et al, 2014;Singhal et al, 2015). In addition, MALDI-TOF MS has been used for species identification of microalgae (Baumeister et al, 2020), microorganisms (Maier et al, 2006) and various metazoan taxa including insects (Feltens et al, 2010), fish (Volta et al, 2012;Shao and Bi, 2020), and calanoid copepods (Riccardi et al, 2012;Laakmann et al, 2013;Bode et al, 2017). Previous studies with several taxa as study subjects confirmed that MALDI-TOF MS is a reliable and fast alternative method for species identification.…”

Plant Single-Cell Metabolomics—Challenges and Perspectives

“…Therefore, gaining an understanding of the physiological status of phytoplankton cells holds excellent promises for environmental research. Besides, chemotyping of microalgae via pipelines that utilize live single-cell MS is currently exploited for taxonomic identification [99]. Despite the broad applications in environmental research, initial studies on algal metabolomics mostly focused on the model organism Chlamydomonas reinhardtii because of applications in the biotechnology industry and biofuel production [100].…”