Untargeted exudate analysis
Rood exudate composition was investigated trough an ultra-high-pressure
liquid chromatography (UHPLC) coupled to a quadrupole-time-of-flight
(QTOF) mass spectrometry as reported by Astolfi et al. (2020)
with minor modifications. Briefly, a 1290 series LC system equipped with
a binary pump, a JetStream Electrospray source and a G6550 iFunnel QTOF
mass spectrometer (Agilent technologies, Santa Clara, CA, USA) working
in positive SCAN mode (100–1200 m/z range) was used. The
chromatographic separation was achieved by a reverse phase Agilent PFP
column (2.0 × 100 mm, 3 μm) using a gradient of water and methanol (from
6% to 94% organic phase in 33 min, flow rate 200 μl
min-1). Quality control pooled samples (QCs) were also
analyzed in MS/MS mode (data-dependent, 12 precursors per cycle at 1 Hz,
50–1000 m/z, positive polarity, active exclusion after 2 spectra), with
collision energies of 10, 20, and 40 eV as previously reported
(García-Pérez, Miras-Moreno, Lucini & Gallego 2021).
Compound annotation was carried out according to Astolfi et al.(2020) from raw mass features, using the software Profinder B.07 (from
Agilent Technologies, Santa Clara, CA, USA) according to the
‘find-by-formula’ algorithm following mass and retention time alignment.
To this aim, the whole isotope pattern (monoisotopic mass, isotopic
spacing and isotopic ratio) was used. A custom database produced by
combining compounds exported from PlantCyc 9.6 (Plant Metabolic Network,
http://www.plantcyc.org; accessed April 2017), Phenol-Explorer 3.6
(http://www.phenol-explorer.eu; accessed April 2017), as well as
information on compounds extracted from the literature and that might be
present in root exudates was used as a reference for annotation (5-ppm
mass accuracy tolerance). The annotation level corresponded to Level 2
of accuracy (putatively annotated compounds) as set out by the COSMOS
Metabolomics Standards Initiative (http://cosmos-fp7.eu/msi). Only those
compounds identified within 75% of replications within at least one
treatment were retained.
The MS-DIAL 4.24 software provided a higher degree of confidence in
annotation, based on MS/MS spectra of QCs to confirm features obtained
by Profinder B.07. (Tsugawa et al. 2015). To this aim, publicly
available built-in MS/MS experimental spectra (Mass Bank of North
America) were used.