SE124:/S1/M1/D1

Sample Set Information

ID	TSE3
Title	Integrated strategy for unknown EI-MS identification using quality control calibration curve, multivariate analysis, EI-MS spectral database, and retention index prediction
Description	Compound identification using unknown electron ionization (EI) mass spectra in gas chromatography coupled with mass spectrometry (GC-MS) is challenging in untargeted metabolomics, natural product chemistry, or exposome research. While the total count of EI-MS records included in publicly or commercially available databases is over 900 000, efficient use of this huge database has not been achieved in metabolomics. Therefore, we proposed a "four-step" strategy for the identification of biologically significant metabolites using an integrated cheminformatics approach: (i) quality control calibration curve to reduce background noise, (ii) variable selection by hypothesis testing in principal component analysis for the efficient selection of target peaks, (iii) searching the EI-MS spectral database, and (iv) retention index (RI) filtering in combination with RI predictions. In this study, the new MS-FINDER spectral search engine was developed and utilized for searching EI-MS databases using mass spectral similarity with the evaluation of false discovery rate. Moreover, in silico derivatization software, MetaboloDerivatizer, was developed to calculate the chemical properties of derivative compounds, and all retention indexes in EI-MS databases were predicted using a simple mathematical model. The strategy was showcased in the identification of three novel metabolites (butane-1,2,3-triol, 3-deoxyglucosone, and palatinitol) in Chinese medicine Senkyu for quality assessment, as validated using authentic standard compounds. All tools and curated public EI-MS databases are freely available in the 'Computational MS-based metabolomics' section of the RIKEN PRIMe Web site ( http://prime.psc.riken.jp ).
Authors	Teruko Matsuo, Hiroshi Tsugawa, Hiromi Miyagawa, Eiichiro Fukusaki
Reference	Matsuo et al. Analytical Chemistry (2017) 89(12):6766–6773
Comment

PubMed

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

Sample Information

ID	S1
Title	Senkyu
Organism - Scientific Name	Cnidium officinate Makino or Ligusticum chuanxiong Hort
Organism - ID	Cnidium officianate：NCBI taxonomy 54711 　Ligusticum chuanxiong：NCBI taxonomy 49555
Compound - ID
Compound - Source
Preparation	Six types of Chinese medicine Senkyu were supplied by Tochimoto Tenkaido Co. Ltd. (Osaka, Japan), and their origins were defined by species (Cnidium officinate Makino or Ligusticum chuanxiong Hort.), cultivation area (Japan or China), and manufacturing process (with or without steaming), as summarized in Supporting Information Table S1.
Sample Preparation Details ID
Comment

Table S1. Details of Chinese medicine Senkyu used in this research

Species	Cultivation area	Process	Abbreviation
Cnidium officinale Makino	Japan	Steaming and dry	CJSD
Cnidium officinale Makino	Japan	Dry	CJD
Cnidium officinale Makino	China	Steaming and dry	CCSD
Cnidium officinale Makino	China	Dry	CCD
Ligusticum chuanxiong Hort	China	Steaming and dry	LCSD
Ligusticum chuanxiong Hort	China	Dry	LCD

Analytical Method Information

ID	M1
Title	EI and GC-MS (Quadrupole)
Method Details ID	MS1
Sample Amount	1 μL
Comment

Analytical Method Details Information

ID	MS1
Title	EI and GC-MS (Quadrupole)
Instrument	GCMS-QP2010 Ultra, Shimazu
Instrument Type
Ionization	EI
Ion Mode	Positive
Description	Extraction from sample A 10 mg sample of each dried root was weighed and homogenized in a 2-mL Eppendorf tube and 1,000 μL of a single-phase mixture of methanol/water/chloroform (5:2:2, v/v/v) containing an internal standard (ribitol, 0.4 mg/mL H2O) was added for metabolite extraction.The extraction was carried out at room temperature using mixer mills (MM 301, Verder Scientific Co. Ltd., Haan, German) at 20 Hz for 5 min.After centrifugation at 16000 ×g for 3 min at 4°C, 800 μL of the supernatant was transferred to a new 1.5-mL Eppendorf tube, to which 400 μL of distilled water was added and vortexed for 10 s. After centrifugation at 16000 ×g for 3 min at 4 °C, 400 μL of the upper layer containing hydrophilic metabolites was transferred to a new 1.5-mL Eppendorf tube, and then freeze-dried (Taitec model VD-800F freeze dryer, Taitec Co. Ltd., Saitama, Japan). For quality control samples, 10 mg of each sample was added into a 15-mL tube (total 360 mg; six biological samples and six biological replicates) and then divided into six 2-mL Eppendorf tubes such that they ontained 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, and 60 mg, respectively. An empty tube was also prepared, labeled as 0 mg QC, for monitoring background effects. Metabolite extraction of these dilution series was performed sing the procedure described above. For oximation, 50 μL of methoxyamine hydrochloride in pyridine (20 mg/mL) was added and incubated at 30 °C for 90 min. For silylation, 50 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was added and ncubated at 37 °C for 30 min. The mixture was centrifuged at 16000 ×g for 5 min at 20 °C and a 50-μL aliquot of the resultant supernatant was transferred into GC–MS vial. Each sample (1 μL) was injected into a GCMS-QP2010 Ultra instrument (Shimadzu Co., Kyoto, Japan) in split mode (25/1, v/v). Each diluted QC sample was analyzed four times from the same GC–MS vial. Analytical conditions were optimized as described in a previous study.14 The column was a fused silica capillary column (30 m × 0.25 mm i.d.) coated with 0.25 μm CP-SIL 8 CB low bleed/MS (Agilent Technologies Japan, Ltd., California, USA). The front inlet temperaturewas 230 °C, the helium gas flow rate was 1 mL/min, and the column temperature was held at 80 °C for 2 min isothermally, then raised by 15 °C/min to 330 °C, and finally held for 6 min isothermally. The transfer line and ion source temperatures were 250 °C and 200 °C, respectively. Twenty scans per second were recorded over the mass range 85–500 m/z.
Comment_of_details

Data Analysis Information

ID	D1
Title	Data Processing
Data Analysis Details ID	DS1
Recommended decimal places of m/z
Comment

Data Analysis Details Information

ID	DS1
Title	Data Processing
Description	NetCDF format files exported from GCMSsolutions (Shimadzu Co., Kyoto, Japan) were converted to Analysis Base Framework (ABF) format files using a free ABF file converter (http://www.reifycs.com/AbfConverter/index.html). MS-DIAL (version 2.48) software was downloaded from the RIKEN PRIMe Web site and used for data processing of the GC–MS data set. The parameters were set as follows: smoothing level, 3; minimum peak height, 2000; average peak width, 20; with default parameters used for the others. The MSP format file (EI–MS reference library) was created using our in-house database and can also be downloaded from RIKEN PRIMe Web site (entitled Osaka Univ. DB). Note that the ranking of structure candidates was based on mass spectral similarity, which was the total score of dot product, reverse dot product, and existence percentage of fragment ions (weighted 2:2:1, respectively) in combination with RI similarity. Details of their mathematical functions followed our previous report. After automatic data processing was finished, the identification results were manually curated with the MS-DIAL graphical user interface by a GC–MS expert, where false positive identification results were changed to “unknown”. A total of 1975 chromatographic peaks (labeled as aligned spots) were created, comprising 127 identified and 1848 unknown peaks (Supporting Information Table S2). All GC–MS data files can be downloaded from the RIKEN Dropmet Web site (http://prime.psc.riken.jp/?action=drop_index).
Comment_of_details

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