SE56:/S01/M01/D01

Sample Set Information

ID	SE56
Title	MS/MS spectral tag-based annotation of non-targeted profile of plant secondary metabolites
Description	We obtained structural information for the detected peaks in the metabolic profile data without performing additional MS/MS analysis; this was achieved by searching for the corresponding MS2T accession in the library. In the case of metabolic profile data for Arabidopsis tissues containing more than 1000 peaks, approximately 50% of the peaks were tagged by MS2Ts, and 90 peaks were identified or tentatively annotated with metabolite information by searching the metabolite databases and manually interpreting the MS2Ts. A comparison of metabolic profiles among the Arabidopsis tissues revealed that many unknown metabolites accumulated in a tissue-specific manner, some of which were deduced to be unusual Arabidopsis metabolites based on the MS2T data. Candidate genes responsible for these biosyntheses could be predicted by projecting the results to the transcriptome data. The method was also used for metabolic phenotyping of a subset of Ds transposon-inserted lines of Arabidopsis, resulting in clarification of the functions of reported genes involved in glycosylation of flavonoids. Thus, non-targeted metabolic profiling analysis using MS2T annotation methods could prove to be useful for investigating novel functions of secondary metabolites in plants.
Authors	Fumio Matsuda, Keiko Yonekura-Sakakibara, Rie Niida, Takashi Kuromori, Kazuo Shinozaki, Kazuki Saito
Reference	Matsuda F et al. (2009) The Plant Journal 57: 555-577
Comment

The original paper is available at The Plant Journal.

The raw data files are available at DROP Met web site in PRIMe database of RIKEN.

Sample Information

ID	S01
Title	Arabidopsis thaliana (Col-0)
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation
Sample Preparation Details ID	SS01
Comment

Sample Preparation Details Information

ID	SS01
Title	Plant materials
Description	Seedlings of Arabidopsis thaliana (Col-0) were grown in pots containing soil at 20℃ with a 16 h daily photoperiod. Six weeks after germination, the 12th or 13th expanded rosette leaves (rosette leaf), the 1st and 2nd expanded cauline leaves (cauline leaf), the upper part of the inflorescence (inflorescence), and first internode tissues (stem) were collected from eight individual Arabidopsis plants at stage 6.3 (Boyes et al., 2001) and stored at -80℃ until use. For metabolic phenotyping of Ds transposon insertion lines (Kuromori et al., 2004, 2006), 60 lines of homozygous seeds were grown on the half-strength MS medium plates at 20℃ with a 16 h daily photoperiod. Two weeks after germination, whole tissues of 20 seedlings were collected, weighed, and stored at -80℃.
Comment_of_details

Kuromori et al. 2006 (PubMed)

Analytical Method Information

ID	M01
Title	Metabolic profiling analysis using LC-ESI-MS
Method Details ID	MS01
Sample Amount	2 μl
Comment

Analytical Method Details Information

ID	MS01
Title	Metabolic profiling analysis using LC-ESI-MS
Instrument	Waters Acquity UPLC system and Waters Q-Tof Premie
Instrument Type	UPLC-QTOF-MS
Ionization	ESI
Ion Mode	Negative
Description	The frozen tissues were homogenized in five volumes of 80% aqueous methanol containing 0.5 mg l)1 lidocaine and D-camphor sulfonic acid (Tokyo Kasei) using a mixer mill (MM 300, Retsch, http://www.retsch.com) with a zirconia bead for 6 min at 20 Hz. Following centrifugation of 15 000 g for 10 min and filtration. <LC-ESI-MS> The sample extracts (2 μl) were analyzed using an LC-MS system equipped with an electrospray ionization (ESI) interface (HPLC, Waters Acquity UPLC system; MS, Waters Q-Tof Premier, http://www.waters.com). The analytical conditions were as follows. HPLC: column, Acquity bridged ethyl hybrid (BEH) C18 (pore size 1.7 μm, length 2.1 · 100 mm, Waters); solvent system, acetonitrile (0.1% formic acid):water (0.1% formic acid); gradient program,1 : 99 v/v at 0 min, 1 : 99 v/v at 0.1 min, 99.5 : 0.5 at 15.5 min, 99.5 : 0.5 at 17.0 min, 1 : 99 v/v at 17.1 min and 1 : 99 at 20 min; flow rate, 0.3 ml min-1; temperature, 38℃; MS detection: capillary voltage, +3.0 keV; cone voltage, 22.5 V; source temperature, 120℃; desolvation temperature, 450℃; cone gas flow, 50 l h-1; desolvation gas flow, 800 l h-1; collision energy, 2 V; detection mode, scan (m/z 100–2000; dwell time 0.45 sec; interscan delay 0.05 sec, centroid). The scans were repeated for 19.5 min in a single run. The data were recorded using MASSLYNX version 4.1 software (Waters). <MS2T data acquisition by LC-Q-TOF-MS> The sample extracts prepared by the method above (2 μl) were subjected to the same LC-Q-TOF-MS system operated under the same conditions mentioned above, except for the following changes: gradient program, 1 : 99 v/v at 0 min, 1 : 99 v/v at 0.2 min, 99.5 : 0.5 at 31 min, 99.5 : 0.5 at 34.0 min, 1 : 99 v/v at 34.2 min and 1 : 99 at 40 min; flow rate 0.15 ml min-1; survey detection mode for MS detection. In this mode, following acquisition of the MS spectrum (m/z 100–1000; dwell time 0.45 sec, inter-scan delay 0.05 sec), the MS/MS data of the most abundant ions were automatically obtained (m/z 50–1000; dwell time 2.5 sec; inter-scan delay 0.5 sec, data acquisition, centroid mode; collision energy ramped from 5 to 60 V). The mass/charge ratio (m/z) was calibrated using the lock-mass function with leucine enkephalin. The analyses were repeated 25 times by shifting the m/z ranges of the target ion selection window for the MS/MS analysis (m/z 100–160, 130–190, 160–220 … 880–940, 940–1000).The data were converted into ASCII format using DataBridge (Waters). The information in each MS/MS spectrum was formatted to the MS2T libraries using in-house Perl scripts. Low-intensity signals of fewer than 5 counts/sec were discarded in this process. The original retention time values were divided by two to compensate for the difference in peak elution conditions.
Comment_of_details

The peak data files of this analysis are available at MS2T.

Data Analysis Information

ID	D01
Title	Profiling by MetAlign and MS2T-based peak annotation
Data Analysis Details ID	DS01
Recommended decimal places of m/z	Default
Comment

Data Analysis Details Information

ID	DS01
Title	Profiling by METALIGN and MS2T-based peak annotation
Description	The data matrix was generated from the metabolic profile data using METALIGN software (de Vos et al., 2007) and processed using in-house software written in Perl/Tk (‘N toolbox’, Appendix S3). Detailed methods for processing and interpretation of the MS2T data are described in Appendix S2. The processed data matrix was analyzed using MeV4.0 (TIGR, http://www.tm4.org) Saeed et al., 2003, 2006).
Comment_of_details

The peak data files of this analysis are available at MS2T.