SE127:/S1/M4/D1

Sample Set Information

ID	TSE6
Title	MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis
Description	Data-independent acquisition (DIA) in liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) provides comprehensive untargeted acquisition of molecular data. We provide an open-source software pipeline, which we call MS-DIAL, for DIA-based identification and quantification of small molecules by mass spectral deconvolution. For a reversed-phase LC-MS/MS analysis of nine algal strains, MS-DIAL using an enriched LipidBlast library identified 1,023 lipid compounds, highlighting the chemotaxonomic relationships between the algal strains.
Authors	Hiroshi Tsugawa, Tomas Cajka, Tobias Kind, Yan Ma, Brendan Higgins, Kazutaka Ikeda, Mitsuhiro Kanazawa, Jean VanderGheynst, Oliver Fiehn & Masanori Arita
Reference	Tsugawa et al. (2015) Nature Methods 12(6):523–526
Comment

PubMed

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

Sample Information

ID	S1
Title	Chlamydomonas reinhardtii
Organism - Scientific Name	Chlamydomonas reinhardtii
Organism - ID	NCBI taxonomy 3055
Compound - ID
Compound - Source
Preparation	The cultivation procedure of Chlamydomonas reinhardtii followed our previous report. The C. reinhardtii CC125 strain was streaked out from cryopreserved stock and cultivated in 75 mL TAP medium in 125 mL shake flasks at 25 °C under constant illumination with cool-white fluorescent bulbs at a fluence rate of 70 µmol m−2 s−1 and with continuous stirring (100 rpm). Four independent cultures were used for this study.
Sample Preparation Details ID	SS1
Comment

Sample Preparation Details Information

ID	SS1
Title	The quenching procedure of algae strains.
Description	One mL cell suspensions were injected into 1 mL of –80 °C cold quenching solution composed of 70% methanol in water, centrifuged at 12,000 g for 2 min, and pellets were lyophilized and stored at −80 °C until further analysis. The same quenching procedure was used for all algae strains.
Comment_of_details

Analytical Method Information

ID	M4
Title	IDA, Negative ion mode
Method Details ID	MS1
Sample Amount
Comment

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

Analytical Method Details Information

ID	MS1
Title	UPLC-Q-TOF Method
Instrument	Agilent 1290 system(UPLC) + AB Sciex TripleTOF 5600+ system (QTOF-MS)
Instrument Type	UPLC-QTOF-MS
Ionization	APCI
Ion Mode	Negative/Positive
Description	Extraction from sample For hydrophilic interaction chromatography (HILIC)-MS/MS analysis of pharmaceutical agents present in a human plasma sample, all procedures for the metabolite extraction were kept on ice. 30 μL of human plasma was added to 1,000 μL cold mix-solvent (acetonitrile/isopropanol/water, 3:3:2, v/v/v) on ice, then vortexed for 10 s and shaken for 5 min at 4 °C using the Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments). After 2 min centrifugation at 14,000 rcf, 300 μL of the supernatant was transferred to a new 1.5 mL Eppendorf tube and evaporated to dryness in a Labconco Centrivap cold trap concentrator. The dried sample was resuspended with 60 μL (80% acetonitrile in water) including 0.038 μg/mL choline-D9, 0.050 μg/mL TMAO-D9, 0.020 μg/mL betaine-D9, 10.0 μg/mL glutamine-D5, and 1.48 μg/mL arginine-15N2 and centrifuged for 5 min at 16,000 rcf. The 50 μL aliquot was transferred to a glass amber vial (National Scientific) with a micro-insert (Supelco). For lipid profiling, all samples for the metabolite extraction were kept on ice and performed as described previously 33. 225μL of MeOH including 1.64 μg/mL PE (17:0/17:0), 6.55 μg/mL PG (17:0/17:0), 1.10 μg/mL PC (17:0/0:0), 0.24 μg/mL sphingosine (d17:1), 0.55 μg/mL ceramide (d18:1/17:0), 0.44 μg/mL SM (d18:1/17:0), 54.5 μg/mL palmitic acid-D3, 0.44 μg/mL PC (12:0/13:0), 22.7 μg/mL cholesterol-D7, 0.27 μg/mL TAG (17:0/17:1/17:0), 2.18 μg/mL DAG (12:0/12:0/0:0), 13.1 μg/mL DAG (18:1/2:0/0:0), 4.36 μg/mL MAG (17:0/0:0/0:0) and 0.55 μg/mL PE (17:1/0:0) were added to each dried algae on ice and vortexed for 10 s. Then, the MTBE including 21.8 μg/mL cholesteryl ester (22:1) was added on ice and vortexed for 10 s. After shaking for 6 min at 4 °C in the orbital mixer, 188 μL water was added and vortexed for 20 s. After centrifugation for 2 min at 14,000 rcf, 350 μL of the supernatant was transferred to a new 1.5 mL Eppendorf tube and evaporated to dryness in the Labconco Centrivap cold trap concentrator. The dried sample was resuspended in 108.6 μL MeOH:toluene 90:10 (v/v) with CUDA (12-[[(cyclohexylamino)carbonyl]amino]-dodecanoic acid, 50 ng/mL). After vortexing for 20 s, each sample was sonicated for 5 min at room temperature. After centrifugation for 2 min at 16,000 rcf, 50 μL of the supernatant was transferred to a glass amber vial with micro-insert. The C. reinhardtii, C. sorokiniana and C. variabilis samples were diluted by adding 50 μL of MeOH:toluene 90:10 (v/v). Moreover, the E. gracilis sample was diluted by adding 200 μL of MeOH:toluene 90:10 (v/v). The liquid chromatography system consisted of an Agilent 1290 system (Agilent Technologies Inc.) with a pump (G4220A), a column oven (G1316C), and an autosampler (G4226A). For hydrophilic metabolite analysis, mobile phase A was 10 mM ammonium formate with 0.125 % formic acid in water; mobile phase B was 95:5 acetonitrile:water (v/v) with 10 mM ammonium formate and 0.125 % formic acid. An Acquity UPLC BEH Amide column (150×2.1 mm; 1.7 µm) coupled to a VanGuard BEH Amide pre-column (5×2.1 mm; 1.7 µm) (Waters; Milford, MA, USA) was used. The gradient was 0 min, 100% B; 2 min, 100% B; 7.7 min, 70% B; 9.5 min, 40% B; 10.3 min, 30% B; 12.8 min, 100% B; 16.8 min, 100% B. The column flow rate was 0.4 mL/min, autosampler temperature was 4 °C, injection volume was 2 µL, and column temperature was 45 °C. For lipid analysis, mobile phase A was 60:40 acetonitrile:water (v/v) with 10 mM ammonium formate and 0.1% formic acid; mobile phase B was 90:10 isopropanol:acetonitrile (v/v) with 10 mM ammonium formate and 0.1% formic acid. The lipidomic LC method utilized an Acquity UPLC charged-surface hybrid (CSH) C18 column (100×2.1 mm; 1.7 µm) coupled to an Acquity CSH C18 VanGuard pre-column (5×2.1 mm; 1.7 µm) (Waters; Milford, MA, USA). The gradient was 0 min, 15% B; 2 min, 30% B; 2.5 min, 48% B; 11 min, 82% B, 11.5 min, 99% B; 12 min, 99% B; 12.1 min, 15% B; 15 min, 15% B. The column flow rate was 0.6 mL/min, autosampler temperature was 4 °C, injection volume was 3 µL in positive mode and 5 µL in negative mode, and column temperature was 65 °C. Mass spectrometry was performed on an AB Sciex TripleTOF 5600+ system (Q-TOF) equipped with a DuoSpray ion source. All analyses were performed at the high sensitivity mode for both TOF MS and product ion scan. The mass calibration was automatically performed every 10 injections using an APCI positive/negative calibration solution via a calibration delivery system (CDS). For hydrophilic interaction chromatography analysis, SWATH (sequential window acquisition of all theoretical mass spectra) acquisition with positive ion mode was used as the data independent acquisition system. The SWATH parameters were MS1 accumulation time, 50 ms; MS2 accumulation time, 30 ms; collision energy, 45 V; collision energy spread, 15 V; cycle time, 640 ms; Q1 window, 25 Da; mass range, m/z 50–500. The other parameters were curtain gas, 35; ion source gas 1, 50; ion source gas 2, 50; temperature, 300 °C; ion spray voltage floating, 4.5 kV; declustering potential, 100 V; RF transmission, m/z 40: 33%, m/z 120: 33%, and m/z 390: 34%. For lipid analysis, six different methods were used; DDA (data-dependent acquisition) with positive ion mode, DDA with negative ion mode, SWATH acquisition (Q1 window, 21 Da) with positive ion mode, SWATH acquisition (Q1 window, 21 Da) with negative ion mode, SWATH acquisition (Q1 window, 65 Da) with positive ion mode, and SWATH acquisition (Q1 window, 65 Da). The common parameters in both SWATH/DDA and positive/negative ion mode were collision energy, 45 V; collision energy spread, 15 V; mass range, m/z 100–1250; curtain gas, 35; ion source gas 1, 60; ion source gas 2, 60; temperature, 350 °C; declustering potential, 80 V; RF transmission, m/z 80: 50%, m/z 200: 50%. The ion spray voltage floating of positive/negative ion mode were +5.5/–4.5 kV, respectively. The DDA parameters in both positive and negative ion modes were MS1 accumulation time, 100 ms; MS2 accumulation time, 50 ms; cycle time, 650 ms; dependent product ion scan number, 10; intensity threshold, 500; exclusion time of precursor ion, 5 s; mass tolerance, 20 mDa; ignore peaks, within 6 Da; dynamic background subtraction, TRUE. The SWATH parameters of 21/65 Da Q1 window were MS1 accumulation time, 100/50 ms; MS2 accumulation time, 10/30 ms; cycle time, 731/640 ms; Q1 window, 21/65 Da.
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	MS-DIAL software and data processing parameters
Description	MS-DIAL is available in Windows OS (.NET Framework 4.0 or later; RAM: 4.0 GB or more). Its source code was written in the C# language with the windows presentation foundation (WPF) to develop the graphical user interface. The main source code such as peak detection, peak spotting, and MS2Dec algorithm is downloadable at http://prime.psc.riken.jp/. The data processing parameter of MS-DIAL utilized in this study are described in Supplementary Table 4.
Comment_of_details

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