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2.1 MS/MS spectra<br />
MassBank dat … 2.1 MS/MS spectra<br />
MassBank data with fragment assignment information (Fragment ion and neutral loss matrix) were obtained from Metabolime.jp (http://metabolomics.jp/wiki/Index:MassBank) (Arita and Suwa 2008; Horai et al. 2010). All procedures were based on in-house scripts written in Perl 5.12. Similarity networks were visualized by Cytoscape 3.2.1 (Shannon et al. 2003). All MS/MS spectral libraries obtained from various tissues of Arabidopsis (ATH06p–ATH014p) (Matsuda et al. 2010) and rice cultivars (OSA06p–OSA014p) (Matsuda et al. 2012) were downloaded from the PRIME webpage (http://prime.psc.riken.jp/, Supplementary Table S1) (Sakurai et al. 2013). Each entry in the MS/MS spectral library has a unique ID such as ATH14p11717 (Matsuda et al. 2009). For visualization and analysis of these MS/MS spectra, (1) please go to the MS2T section of the RIKEN PRIMe web page (http://prime.psc.riken.jp/lcms/ms2tview/ms2tview.html), (2) input IDs (e.g., ATH14p11717) into the “MS/MS spectrum tag(MS2T) accession codes” field, and (3) press the Start button. (4) A list of MS/MS spectra will appear. (5) Press the Detail:[ATH14p11717] button for detailed analysis of the MS/MS spectra. The “Molecular formula search” function is also available.<br />
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2.2 Conversion of MS/MS spectra to MS/MS strings<br />
MS/MS strings were generated from the MS/MS spectra by the following procedure. For each entry in the MS/MS spectral libraries,<br />
1.
The entry was discarded when intensity of the most intense fragment (base peak) was less than 30 counts per second.<br />
2.
The fragments whose intensity was less than that of the 12th most intense signal were ignored. Fragment signals of weak intensity (less than 1.0 % of the base peak) were also ignored.<br />
3.
The chemical formula of precursors was assigned according to the seven golden rules (Kind and Fiehn 2007).<br />
3.1
The chemical formulas including CHONS atoms with 0 ≤ N ≤ 5 and 0 ≤ S ≤ 3 were considered. All formulas within the threshold (10 mDa [m/z < 500] and 20 ppm [m/z > 500]) were used to construct MS/MS strings.<br />
3.2
For each chemical formula of a precursor, the chemical formulas of all fragments were deduced according to the seven golden rules, with the threshold at 20 ppm. The numbers of atoms in a molecular fragment had to be less than those in the precursor.<br />
3.3
The total intensity of fragment signals whose chemical formulas were successfully elucidated (I selected) was calculated. The chemical formula of a precursor was accepted when I selected ÷ I total was greater than 0.8, where I total indicates total intensity of all fragment signals (Meringer and Schymanski 2013).<br />
4.
An entry without an accepted chemical formula of a precursor was discarded.<br />
5.
All possible MS/MS strings were generated using the following format: [formula of neutral loss 1]:[formula of fragment 1];[formula of neutral loss 2]:[formula of fragment 2];… [formula of neutral loss n]:[formula of fragment n];.<br />
5.1
The threshold for finding a chemical formula of a neutral loss was 15 mDa.<br />
5.2
One neutral loss involving negative atom numbers (such as; C1H-2:) was allowed in 1 MS/MS string.<br />
5.3
One fragment with an integer index of hydrogen deficiency (IHD) was allowed in 1 MS/MS string.<br />
The MS/MS strings that were generated by this procedure were incomplete because steps 5.2 and 5.3 are heuristic conditions designed to restrict a combinatorial explosion of the number of MS/MS strings. All MS/MS string libraries derived from MassBank, Arabidopsis, and rice data were downloaded from the project homepage (http://www-shimizu.ist.osaka-u.ac.jp/hp/en/software/regex.html) and DROP Met in PRIMe website (http://prime.psc.riken.jp/?action=drop_index). ://prime.psc.riken.jp/?action=drop_index).
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