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rsc.li/metallomics Page 1 of 22 Metallomics View Article Online DOI: 10.1039/C7MT00098G 1 2 3 Comprehensive non-target analysis identifies 54 sulphur containing compounds in garlic. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

25 Manuscript 26 27 28 29 30 31 32 33 34 35 Accepted 36 37

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 39 40 41 42 43 44 45 46 Metallomics 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Metallomics Page 2 of 22 View Article Online DOI: 10.1039/C7MT00098G 1 2 3 4 1 Sulphur fertilization influences the sulphur species composition 5 6 2 in Allium sativum: sulphomics using HPLCICPMS/MSESI 7 8 3 MS/MS 9 4 Andrea Raab, Marilena Ronzan, Joerg Feldmann 10 11 5 TESLA (Trace Element Speciation Laboratory), University of Aberdeen, Chemistry, 12 6 Meston Walk, Aberdeen, AB243UE, Scotland, UK 13 14 7 15 16 17 8 Abstract 18 19 9 Garlic ( A. sativum ) contains a large number of small sulphur (S)containing 20 10 metabolites, which are important for its taste and smell and vary with A. sativum 21 11 variety and growth conditions. 22 23 12 This study was designed to investigate the influence of different sulphurfertilization 24 13 regimes on the low molecular weight Sspecies by attempting the first sulphur mass 25 Manuscript 26 14 balance in A. sativum roots and bulbs using HPLCICPMS/MSESIMS/MS. 27 28 15 Species unspecific quantification of acid soluble Scontaining metabolites was 29 16 achieved using HPLCICPMS/MS. For identification of the compounds high 30 31 17 resolution ESIMS (Orbitrap LTQ and qTOF) was used. 32 33 18 The plants contained up to 54 separated sulphurcontaining compounds, which 34 19 constitute about 80 % of the total sulphur present in A. sativum . Roots and bulbs of Accepted 35 20 A.sativum contained the same compounds, but not necessarily the same amounts 36 21 and proportions. The Scontaining metabolites in the roots reacted more sensitive to 37

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 22 manipulations of sulphur fertilization than those compounds in the bulbs. In addition 39 23 to known compounds (eg. γglutamylS1propenylcysteine) we were able to identify 40 24 and partially quantify 31 compounds. Three as yet undescribed Scontaining 41 42 25 compounds were also identified and quantified for the first time. Putative structures 43 26 were assigned to the oxidised forms of S1propenylmercaptoglutathione, S2 44 27 propenylmercaptoglutathione, Sallyl/propenylcontaining PC2 and 2amino3[(2 45 28 carboxypropyl)sulfanyl]propanoic acid. 46 Metallomics 47 29 48 The parallel use of ICPMS/MS as sulphurspecific detector and ESIMS as 49 30 molecular detector simplifies the identification and quantification of sulphur 50 31 containing metabolites without species specific standards. This nontarget analysis 51 32 approach enables a mass balance approach and identifies the occurrence of so far 52 53 33 unidentified organosulphur compounds. The experiments showed that the sulphur 54 34 fertilization regime does not influence sulphurspeciation, but the concentration of 55 35 some Scontaining compounds in roots is depending on the sulphur fertilization. 56 57 36 58 59 60 Page 3 of 22 Metallomics View Article Online DOI: 10.1039/C7MT00098G 1 2 3 37 Significance to Metallomics 4 5 38 Sulphur is not a metal, but the similarity to Se which is featured in the journal should 6 39 make an Sbased study eligible. The multitude of Scontaining metabolites in allium 7 8 40 is difficult to quantify using traditional methods. We developed a species 9 41 independent quantification method coupled with simultaneous identification using 10 42 HPLCICPMS/ESIMS to give a holistic (sulphomic) view on the acid soluble low 11 43 molecular weight Smetabolites. This is in the spirit of nonmetal non target 12 1 13 44 speciation analysis as laid out recently. 14 15 45 16 17 46 Introduction 18 47 19 20 48 A wide variety of Allium sativum (garlic) is cultivated worldwide for use as spice in 21 2 22 49 the kitchen and for their medical properties. Like all alliums garlic contains a variety 23 50 of volatile and nonvolatile sulphur (S) containing metabolites, which are mainly 24 51 responsible for its typical smell and taste. Several sulphur containing phytochemicals

25 Manuscript 52 26 present in A. sativum show at least in vitro medical properties, namely alliin and 3 27 53 allicin, supporting the use of garlic in traditional medicine. Suggestions of health 28 54 benefits resulting from consumption of garlic range from reducing the risk of coronary 29 55 heart disease to anticancer properties.4,5 Allicin, the major volatile Sspecies 30 31 56 produced by crushing garlic and first identified in 1944 by Cavallito, was shown to 32 57 have significant bacteriostatic activity in vitro .2 Clinical trials have, however, so far 33 58 failed to show conclusive evidence for significant health benefits.6 34 Accepted 35 59 Research in sulphurcontaining compounds (Scontaining compounds) of garlic 36 37 60 focuses predominantly on alliin, allicin and some of their major derivatives. The

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 61 presence of some di and tripeptides of the γglutamyl cysteinyl family containing an 39 62 Sallyl or Spropenyl moiety is known. The best known are γglutamylSallylL 40 63 cysteine (GSAC), γglutamylS1propenylLcysteine (GSPC) and γLglutamylS 41 5,7,8 42 64 methylLcysteine (GSMC). Whether they are precursors and / or sulphur storage 43 65 peptides, especially during favourable growing conditions, for the eventual formation 44 66 of alliin and allicin, is one example of what is not known in the biosynthesis of S 45 67 46 containing molecules. Metallomics 47 48 68 The main reasons for this lack of knowledge are the use of unspecific analytical 49 69 techniques for the determination of nonvolatile Scontaining compounds; mainly 50 70 HPLCUV with quantification & identification of compounds by synthetic standards is 51 71 used. Rarely ESIMS is used for identification of Scontaining compounds in garlic. 52 8,7 53 72 Quantification is always done using synthesised standard compounds. When ESI 54 73 MS is used for identification of unknown Scontaining compounds high accuracy not 55 74 only with regard to m/z , but also the isotopic pattern is required. The latter is 56 75 57 important, since the sulphur isotopic pattern is not very different from the isotopic 58 76 pattern contributed by carbon, oxygen, nitrogen and hydrogen present in the 59 60 Metallomics Page 4 of 22 View Article Online DOI: 10.1039/C7MT00098G 1 2 3 77 compound. 32 S constitutes about 95 % of the present sulphur with 33 S contributing 4 78 about 0.8 %, 34 S 4.2% and 36 S 0.02%. Sulphur has also a small massdeficiency, a 5 1 6 79 compound of similar composition (m: 305 g mol ) containing one sulphur atom is 1 7 80 about 0.0905 g mol lighter than a compound containing only C,N,O and H. Both 8 81 mass defect and isotope pattern shift can be applied to identify unknown compounds 9 82 when using highresolution accurate MS instruments.9 But a nontarget analysis with 10 11 83 mass balance approach is not possible using molecular mass spectrometry. 12 13 84 The aim of this feasibility study was to test firstly whether the parallel use of ICP 14 85 MS/MS and ESIMS is advantageous for quantification and identification of S 15 86 containing compounds in roots and bulbs of A. sativum without the use of species 16 17 87 specific standards. Secondly we applied this approach to study the influence of the 18 88 levels of sulphur fertilization on the generation of the different Scontaining 19 89 compounds and calculated a complete sulphur mass balance (sulphomics). 20 21 90 22 23 91 Material and methods 24

25 92 Manuscript 26 93 Chemicals and Standards 27 28 94 MilliQ water (18 M cm, Millipore UK) was used throughout for the preparation of 29 95 every solution except growing of A. sativum . Other chemicals (hydrogen peroxide, 30 96 concentrated nitric acid, cysteine, formic acid) were of at least p.a. quality (all from 31 97 Sigma, UK), methanol was of HPLCgrade (Sigma, UK). The sulphur standard for 32 33 98 total sulphur determination and the germanium solution used as continuous internal 34 99 standard were from High Purity Standards (USA). As certified reference material Accepted 35 100 (CRM) for total S determination RM 8415 (whole egg powder, NIST, Gaithersburg, 36 101 37 USA) and Seronorm urine blank (Sero, Norway) were used. No CRM for sulphur

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 102 speciation was available. 39 40 103 Plants 41 104 A. sativum (single bulb garlic of Chinese origin) was bought in a shop in Aberdeen, 42 105 UK and grown hydroponically for 6 weeks at 19 +/ 1ºC with ambient lighting. Each 43 44 106 bulb was individually grown in a plastic beaker. The plants were fertilized using 45 107 Hoagland’s solution (20 % strength) with the sulphur levels adapted by either 46 108 replacing the sulphur containing salts from Hoaglandsolution by chloride containing Metallomics 47 48 109 salts or by adding increased levels of magnesium sulphate (see details ESI). The 49 110 solutions were replaced 3 times per week. Sulphur levels at which the plants were 50 111 grown for 6 weeks were 0.1, 0.5 and 2 mM sulphate, 20 bulbs were grown on each 51 112 level and 4 or 5 plants randomly selected for sampling in two separate (8 weeks 52 53 113 difference in planting), but otherwise identical experiments. Dry weight (d.w.) of plant 54 114 parts was determined by freeze drying. Roots contained 9.9 ± 0.5 % dry matter and 55 115 bulbs 24 ± 2.7 %. 56 57 58 59 60 Page 5 of 22 Metallomics View Article Online DOI: 10.1039/C7MT00098G 1 2 3 116 Sample preparation 4 117 The plants were harvested after 6 weeks. Their roots rinsed with deionised water 5 6 118 and blotted dry. Roots and shoot were separated from the bulb. Only freshly formed 7 119 (during the 6 weeks of the experiment) bulb tissue was used. Roots and bulb were 8 120 separately grounded to fine powder using liquid nitrogen as soon as separated. 9 121 Material intended for species determination was kept frozen with liquid nitrogen until 10 11 122 extraction with 1 % (v/v) formic acid in water in an ice bath (~ 1ºC) for 15 min (1 g 12 123 plant / 4 mL extraction solution). The extract was centrifuged and the supernatant 13 124 was used immediately for speciation analysis. Formic acid was used to suppress 14 125 15 allinase (EC 4.4.1.4) activity which is irreversibly inhibited at pH below 3.5, as 7 16 126 described by Ichikawa et al . 17 18 127 For total sulphur determination the plant material and reference materials (100 ± 0.1 19 128 mg) was digested using 1 mL nitric acid and 2 mL hydrogen peroxide in a microwave 20 129 oven (Mars5, CEM) for 30 min at 95ºC in unpressurised vessels. The digests were 21 22 130 diluted with water to 50 g before sulphur determination. Extracts prepared for 23 131 speciation analysis were diluted 1 to 50 with 1 % nitric acid for determination of total 24 132 extracted sulphur.

25 Manuscript 26 133 Instrumentation 27 28 134 Species separation 29 135 An Agilent 1100 HPLC or a 1290 HPLC system with cooled autosampler was used 30 136 for separation. The extract was separated using an Agilent Eclipse C18 column (4.6 31 137 32 * 150 mm) with a linear water methanol gradient (both 0.1 % v/v formic acid) in 20 1 33 138 min to 20 % methanol and held for 10 min. The flow rate was 1 mL min , after the 34 139 column the flow was split 1:3 with 1 part introduced into the ICPMS/MS and the rest Accepted 35 140 into the ESIMS (QuickSplit Postcolumn Flow splitter, Analytical Scientific 36 37 141 Instruments, USA). The sample volume was 0.1 mL and the column oven set to

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 142 40ºC. 39 40 143 Quantification and S-specific detection using ICP-MS/MS 41 144 An 8800 Agilent ICPMS/MS was used for all measurements in MS/MS mode using 42 145 oxygen (30 %; ~ 0.3 mL min 1 O ) and hydrogen (1.1 mL min 1 H ) as reaction gases. 43 2 2 44 146 The energy discrimination was set to 7 mV with a wait time of 2 ms, all other 45 147 parameters were optimised daily as required. For total sulphur determination and 46 148 determination of sulphur in the extracts the instrument was used with nickel cones in Metallomics 47 48 149 standard setup. Due to the high concentration, only sulphur isotopes 33 and 34 with 33 + 49 150 mass shift of 16 were measured on m/z 49 and m/z 50 (Q1: m/z 33 and 34, S and 50 151 34 S+, Q2: m/z 49 or 50, 33 S16 O+ and 34 S16 O+). Germanium was used as continuous 51 152 internal standard added online and measure on mass ( m/z 72). 52 53 153 For sulphur speciation analysis the instrument was used in organic mode with Pt 54 55 154 cones, microPFA nebulizer and the addition of 6 % oxygen/argon (20:80) to the 56 155 nebulizer gas. Q1 was set to m/z 32 or 34 while Q2 was set in the mass shift mode 57 156 to m/z 48 ( 32 S16 O+) and 50 (34 S16 O+). Continuous internal standard (germanium) was 58 59 60 Metallomics Page 6 of 22 View Article Online DOI: 10.1039/C7MT00098G 1 2 3 157 added via a Tpiece before the nebulizer and measured on mass ( m/z 72). The 4 158 influence of the methanol gradient on signal intensity was corrected for as described 5 10 6 159 in Amayo et al. Standards used for quantification were prepared fresh every day 7 160 from cysteine in 1 % (v/v) formic acid. For peak integration PeakFit (Jandel 8 161 Scientific) was used. The program was used with Method I Residuals and the 9 162 integration model used was EMG + GMG, baseline setting was 0.1 % linear. Peak 10 11 163 parameters varied during integration were residuals, width and shape. Starting peaks 12 164 were set manually at the signal maximums. The results of five chromatograms for 13 165 experiments 1, respectively 2 were integrated repeatedly at 3 different days 14 166 15 (independently), the peak areas for individual peaks varied on average by 3 to 5 %. 16 167 An example of the results is given in Fig. S32. 17 18 168 Identification of S-species using ESI-MS/MS 19 169 An Orbitrap Discovery (Thermo Scientific) was used for the identification of the 20 170 eluting compounds, when the instrument was coupled to the HPLC in parallel to the 21 11 22 171 ICPMS/MS as described in more detail elsewhere. The instrument was used in 23 172 positive mode with 4.5 kV source voltage at 30.000 resolution in MSmode and a 24 173 scan range from 1001500. One MSMS was measured after each MSspectrum

25 Manuscript 174 when it was triggered (minimum 10000 counts) at a resolution of 7500 in CID mode 26 27 175 (activation Q: 0.25, normalised collision energy: 35, isolation width: 1 m/z , activation 28 176 time: 30, wideband activation). Additionally experiments were run on an Agilent 6200 29 177 series TOF/6500 series QTOF instrument using the same HPLC conditions and 30 31 178 similar ESIconditions with a scan rate of 1.5 Hz, scan range from 100 1000, 32 179 variable CID energy, 3.5 kV source voltage, fragmentor 175 V (± 200 %) and 33 180 reference masses (121.05087 and 922.00979) enabled. The instruments were 34 181 optimised as required. For identification / confirmation of fragmentation patterns Accepted 35 12 13 36 182 MetFusion was used with ChemSpider as database. The molecular formulas 37 183 were accepted as correct when ∆ppm was less than 3 ppm of the theoretical m/z .

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 39 184 Statistical analysis 40 185 All significant levels were tested using SigmaPlot 13.0 One Way ANOVA. Errors are 41 186 always given as standard deviation of nine biological replicates if not mentioned 42 43 187 otherwise. Mintab 17 was used as a platform for chemometric calculations: a) 44 188 unsupervised principal component analysis (PCA) and cluster analysis was 45 189 employed for the 22 identified and quantified low molecular weight Scontaining 46 Metallomics 47 190 metabolites including sulphate from all fertilization stages. 48 49 191 50 51 192 Results and discussion 52 193 53 54 194 Single bulb garlic used in these experiments is an A. sativum variety with 55 195 significantly lower pungency than “normal” multiple clove forming A. sativum 56 57 196 varieties. The term single bulb garlic was for simplicity shortened to garlic throughout 58 197 the paper. 59 60 Page 7 of 22 Metallomics View Article Online DOI: 10.1039/C7MT00098G 1 2 3 198 Total sulphur in garlic roots and freshly formed bulb-tissue 4 199 5 6 200 Contamination of all liquids from environmentally present gaseous Scontaining 7 201 8 compounds is always a risk, leading to elevated sulphur background levels. All 9 202 solutions were therefore not stored any longer than necessary and standards were 10 203 prepared on the same day as the samples and stored under identical conditions. To 11 204 reduce the risk of sulphur contamination, due to the presence of sulphur in the 12 13 205 chemicals used for digestion including the water, the dilution factor of the plant 14 206 digests was kept relative small and the sulphur concentration in the standards 15 207 relative high (up to 0.6 mmol kg 1). To reduce the amount of ions hitting the detector 16 208 16 33 34 17 sulphur was determined via O mass shift on S and S. In RM8415 with a certified 1 1 18 209 value of (5100 ± 500) mg S kg (4713 ± 100) mg S kg (n = 4) was determined, 19 210 Seronorm urine (blank) (658 ± 70) mg S kg 1 (certified: 545 ± 70, n = 3). The 20 211 recovery of the reference materials was between 92 and 121 %. The limit of 21 1 1 22 212 determination was between 10 and 20 mol kg (0.3 – 0.6 mg kg ) sample (n = 5). 23 24 213 Total sulphur concentration in roots was strongly depending on sulphur fertilization (p

25 214 < 0.01). Plants fertilised with 0.1 mM sulphate for 6 weeks containing significantly Manuscript 26 215 less sulphur than plants fertilised with either 0.5 or 2 mM (Table 1). From the growth 27 216 28 behaviour of garlic it can be estimated that at least 0.5 mM bioavailable sulphur are 29 217 required for optimal growth of the roots (details not shown). The sulphur content in 30 218 roots increased linearly over the three tested sulphur levels (r²: 0.91). In contrast to 31 219 the roots there was no significant difference in the sulphur concentration of the newly 32 33 220 formed bulb material (p = 0.236) (Table 1), but the sulphur content of the individual 34 221 bulbs varied significantly within the groups. Montano et al .8 also found a high Accepted 35 222 variability of specific Scontaining compounds in cloves of the same bulb and 36 223 between bulbs (up to 36% depending on A. sativum variety) without determining the 37

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 224 total sulphur content. The same variability is likely to occur for total sulphur since the 39 225 majority of S is present as small acid extractable species. The amount of newly 40 226 formed bulb tissue did not seem to be influenced by the availability of sulphur from 41 42 227 the fertilizer within 6 weeks of growth. 43 44 228 A large proportion of sulphur could be extracted (80.8 ± 14.8) % (n = 54) using 1 % 45 229 formic acid as solvent independent of the amount of sulphur fertilization (Table 1). 46 230 This indicates that the majority of Scontaining compounds present in garlic are small Metallomics 47 231 48 acid soluble Scontaining compounds. About 20 % of the sulphur is not extractable 49 232 by 1 % (v/v) formic acid and might be present as proteinbound sulphur. 50 51 233 Comparing the Sconcentration found here with literature values showed that garlic 52 234 grown to maturity in some field trials contained significantly more sulphur. 15 Sulphur 53 235 content seems to be highly variable and depending on the availability of sulphate in 54 55 236 soil and on A. sativum variety. In a field trial applying different Se and humic acid 56 237 concentrations sulphur concentration between 0.3 and 0.5 % d.w. were found in 57 238 bulbs (96 158 mmol S kg 1 d.w., the sulphur concentration in soil was not 58 59 60 Metallomics Page 8 of 22 View Article Online DOI: 10.1039/C7MT00098G 1 2 3 239 specified). 14 These values are similar to the ones found in the hydroponically grown 4 240 bulbs here. In contrast bulbs of A. sativum L. var. Thermidrome grown under 5 6 241 different S and N regimes in Germany contained roughly 10 to 20 times more 7 242 sulphur at growth stages 2 and 3 depending on fertilization (these stages are 8 243 comparable with our harvested plants). 15 9 10 244 Garlic roots are normally not studied, the ones studied here contained between 2 11 245 and 3.5 times as much S than the bulb. The reason is currently unknown. 12 13 14 246 15 247 Table 1 amount of sulphur determined in root and bulb of garlic plants exposed 16 1 17 248 to variable amounts of sulphate for 6 weeks in mmol S kg d.w. (mean ± standard 18 249 deviation, n per group = 9). 19 20 0.1 mM S 0.5 mM S 2 mM S 21 Root total 202 ± 88a,b 279 ± 71a,c 466 ± 59b,c 22 Root extractable (% 164 ± 71 a 211 ± 45b 353 ± 50a,b 23 extraction efficiency) (81 %) (76 %) (76 %) 24 Root sum 138 ± 53 b 172 ± 89c 383 ± 86 b,c 25 # Manuscript 26 chromatogram (% (84 %) (82 %) (108 %) 27 column recovery) 28 Bulb total 135 ± 25 130 ± 33 133 ± 40 29 Bulb extractable (% 116 ± 42 110 ± 32 116 ± 48 30 extraction efficiency) (86 %) (85 %) (87 %) 31 Bulb sum 97.7 ± 41 90.3 ± 35 (82 100 ± 46 32 chromatogram # (% (84 %) %) (86 %) 33 column recovery) 34 a,b,c 250 : statistically significant difference between groups by One Way ANOVA p < 0.001 Accepted 35 251 # sum chromatogram: sum of all individually integrated peaks 36 252 37

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 253 39 40 254 41 42 43 255 Sulphur-speciation analysis (Sulphomics) 32 16 + 44 256 For speciation analysis sulphur was measured on m/z 48 ( S O ) to increase the 45 257 sensitivity of the ICPMS/MS, since only a quarter of the injected sample was infused 46 258 into the instrument (the rest being directed to the ESIMS). Chromatograms of garlic Metallomics 47 48 259 root and bulb extract separated under the chosen conditions contained around 54 49 260 separated Scontaining peaks, some peaks contained several compounds. The 50 261 sulphur content in each chromatographic peak was quantified using external 51 262 10 52 calibration with correction for the carboneffect as described by Amayo et al. The 53 263 concentration of the detected Scontaining compounds ranged from 0.01 to 100 54 264 mmol S kg 1 d.w. per chromatographic peak as determined by ICPMS/MS. The l.o.d. 55 265 was about 0.005 mmol S kg 1 d.w. The interplant variability was very high 56 57 266 (depending on compound between 10 and 160 % RSD were found, n = 9) and only 58 59 60 Page 9 of 22 Metallomics View Article Online DOI: 10.1039/C7MT00098G 1 2 3 267 with the statistical power of using 9 replicates, differences on the compound level 4 268 became significant between the fertilization stages. 5 6 269 The results of the quantification by ICPMS/MS were compared with literature values 7 8 270 determined by HPLCUV. These values do not necessarily compare, since HPLCUV 9 271 results may not be free of interfering compounds coeluting with the compound in 10 272 question. In none of the publications the possibility of interfering compounds in real 11 273 samples was studied, although due to the complexity of the matrix and the number of 12 13 274 Scontaining compounds (and others) present in extracts coeluting UVactive 14 275 compounds cannot be excluded. These interfering compounds will influence the 15 276 quantification results using HPLCUV with external calibration and also with standard 16 17 277 addition. The comparison of concentrations between the quantification using HPLC 18 278 UV and HPLCICPMS/MS can therefore only be used as a guide. 19 20 279 Set prerequisites for the identification as Scontaining compound: 21 22 280 (i) Signal in the sulphur trace of the ICPMS/MS 23 281 (ii) Manual mining of the ESIMS data at retention time of signal 24 282 (iii) Extraction of EIC of potential m/z within less than 2 ppm error of the 25 Manuscript 26 283 theoretical m/z 27 284 (iv) The shape of the chromatographic peak of the extracted mass charge ratio 28 285 with the shape of the sulphur peak from the ICPMS/MS trace 29 30 286 (v) The potential elemental composition (∆ppm < 3 ppm) and 31 287 (vi) The MS 2 data (when available). 32 33 288 In this way the identification of the major compounds was unambiguous but the 34 289 identification of isomer/diastereomer would need confirmation from e.g. NMR. An Accepted 35 36 290 example is shown in Fig. 1, the Figures for other compounds can be found in the 37 291 electronic supplement (Fig. S1 to S27), while Fig. 2 illustrates the complexity of the 38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 292 data mining showing the EIC for 9 different Scontaining compounds in one 39 293 chromatogram. In Fig. S30 & S31 examples of sulphur traces for 3 root and their 40 41 294 corresponding bulb extracts are shown. Table 2 contains a summary of all identified 42 295 compounds. The identification of minor Scontaining compounds was still difficult, 43 296 especially since Scontaining compounds proved to be very reactive under ESIMS 44 45 297 conditions (eg. Fig. S8). Compounds containing the moiety –S(O)C 3H5 were 46 298 especially reactive. Depending on compound this led to insource fragmentation, in Metallomics 47 299 source oxidation/reduction or multimer formation impeding the identification of the 48 300 compound. The majority of secondary sulphur metabolites in A. sativum are relatives 49 50 301 of cysteine or γglutamylcysteine often containing an additional sulphur moiety 51 302 (related to alliin). Methionine containing compounds were a minority. A number of 52 303 these compounds were present in different isoforms and / or diastereomers. 53 54 304 The major Scontaining compounds in garlic were hydrophilic compounds (including 55 56 305 sulphate) and the alliinvariants (cycloalliin, methiin, alliin, isoalliin and propiin), 57 306 together containing between 11 and 26 % of the total sulphur in root (Table S1) and 58 59 60 Metallomics Page 10 of 22 View Article Online DOI: 10.1039/C7MT00098G 1 2 3 307 20 % in bulb. The sulphate, alliin, isoalliin and propiin content increased significantly 4 308 between low and high (p < 0.05) or medium and high exposure groups (p < 0.05) in 5 6 309 roots, but not in bulb (Table S1). The interplant variability in bulb material was 7 310 higher than in roots, which may contribute to the fact that no influence of sulphur 8 311 fertilization in bulb for any compound was found which confirmed the finding by 9 312 Montano and coworkers . 8 10 11 313 Major S-containing compounds cited in the literature and quantified 12 13 14 314 Alliin and its isomers are the precursors for allicin and other thiosulfinates, which are 15 315 the major compounds responsible for the typical garlic taste and smell. Their 16 316 quantification therefore is of major interest to producers of garlic containing food and 17 317 food supplements. Determination by HPLCUV (the standard method used in the 18 1 19 318 literature) results in highly variable alliin (59 and 298 mmol alliin kg d.w.) content in 20 319 bulbs. 8,15,16 The variation of this dominant species results most likely from different 21 320 garlic varieties and culture conditions. The content also varied with age of the plant. 15 22 321 23 The garlic grown in our experiment is a very mild variety and this also showed in the 24 322 alliin content, which was significantly lower than literature values both in bulb (21 1 25 323 mmol alliin kg d.w., Table S1) and root. The alliin content in root was depending on Manuscript 26 324 the level of Sfertilization (between 14 and 79 mmol alliin kg 1 d.w.). The isoalliin 27 1 28 325 content in bulbs (4 mmol kg d.w., Table S1) was within the range mentioned in the 29 326 literature. 7,8 Cycloalliin eluting very early on was not cleanly separated from other S 30 327 containing compounds(Table S13). 31 32 328 The published concentrations of the γglutamyl relatives of aliin and isoalliin (γ 33 34 329 glutamylSallylcysteine (GSAC) and γglutamylS1propenylcysteine (GSPC)) 1 7,8,16 Accepted 35 330 (structures Table S4) range from 7.5 to 224 mmol kg d.w. for GSAC and for 36 331 GSPC from 27 to 312 mmol kg 1 d.w.7,8,16 The content of both compounds 37 332 determined here via sulphur was significantly lower (Table S4). The content of GSPC 38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 39 333 was higher than that of GSAC both in bulb and root. Roots contained slightly more 40 334 GSAC than bulb, but less GSPC than bulbs. GSPC content in root was depending 41 335 on Sfertilization. The oxidised isomers of GSAC (GSAC(O)) and GSPC (GSPC(O)) 42 336 43 eluted significantly earlier than the parent compounds. Both compounds have been 17 44 337 reported as being present in garlic by Yamazaki et al . They were quantified by 45 338 Hughes et al. 18 studying changes in GSAC(O) and GSPC(O) in garlic bulbs and 46 339 cloves due to storage conditions with concentrations found for GSAC(O) between Metallomics 47 1 1 48 340 6.2 and 68 mmol kg and between 27 and 202 mmol kg for GSPC(O) with no 18 49 341 indication of whether it was determined on a dry or fresh weight basis. In our study 50 342 the amount of GSPC(O) in bulbs was lower than that of GSAC(O) in contrast to 51 343 Hughes findings (Table S5). Roots contained similar amounts of both compounds at 52 53 344 the low and medium fertilization level, but significantly more GSAC(O) than 54 345 GSPC(O) at the high level (with a very high interplant variability in all cases. In all 55 346 groups the concentrations of the oxidised forms in roots were by more than a factor 56 57 347 of 10 higher than the reduced forms GSAC and GSPC (Table S4 and S5). 58 59 60 Page 11 of 22 Metallomics View Article Online DOI: 10.1039/C7MT00098G 1 2 3 348 The content of the methiin relative γglutamylSmethylcysteine (GSMC) showed a 4 349 sulphurfertilization dependency in roots (up to ca. 1.7 mmol kg 1 d.w., Table S6), 5 1 6 350 whereas the content in bulbs was about 0.08mmol GSMC kg d.w. (Table S6), low 7 351 when compared to published values. Literature values for GSMC for garlic range 8 352 between 0.38 and 144 mmol kg 1 d.w. 7,8 9 10 353 Reduced glutathione (MS 2 by Orbitrap, but not qTOF) coeluted with propiin and 11 354 methionine showing a very small signal in ESMS. Oxidised glutathione (GSSG) 12 13 355 eluted without coelution of any other Scontaining compound. Its concentration was 14 356 not influenced by the Sfertilization regime. Bulbs contained about 0.09 mmol kg 1 15 357 d.w. GSSG (equiv. 0.18 mmol reduced GSH) and roots between 0.17 and 0.27 mmol 16 1 1 17 358 GSSG kg d.w. (equiv. 0.35 to 0.55 mmol kg d.w. reduced GSH) (Table S7). Since 18 359 the signal for reduced GSH also contained coeluting propiin and methionine (~ 0.9 19 360 mM S kg 1 d.w. in bulb and between 1.1 and 3.2 mmol S kg 1 d.w. in root), no 20 361 quantification of reduced GSH was possible. However, the values for GSSG 21 1 22 362 compared well with the 0.6 to 1.9 mmol kg d.w. as total GSH in bulbs after 23 363 reduction as reported by Bloem et al .15 The fact that GSSG is the more dominant 24 364 form of GSH is slightly unusual, but not an artefact of sample preparation since

25 Manuscript 26 365 dissolving reduced GSH in 1 % formic acid does not lead to GSSG formation (data 27 366 not shown). Therefore GSSG must be naturally the more dominant of the two forms. 28 29 367 30 31 368 Compounds so far not quantified in the literature 32 33 369 The content of propiin, the coeluting methionine and reduced glutathione increased 34 1 370 in roots with Sfertilization (1.2 to 3.2 mmol sulphur kg d.w.), whereas bulbs Accepted 35 1 36 371 contained on average 1 mmol S kg d.w. (Table S13) at this retention time. None of 37 372 the compounds was as yet quantified in garlic. Assuming similar sensitivity in ESI 38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 373 MS for all three compounds (likely due to their similarity) propiin was the dominant 39 374 compound. 40 41 42 375 The Sallylcysteine (SAC, deoxyalliin) content in bulbs was slightly lower than in 43 376 roots and did not depend on the Sfertiliser regime (Table S3), possibly due to high 44 377 interplant variability. SAC identification by extracted ionchromatogram was difficult, 45 378 since a whole host of other compounds showed insource fragments at m/z 46 Metallomics 47 379 162.0583 among these were S1propenylmercaptoglutathione, S2 48 380 propenylmercaptoglutathione and Spropylmercaptoglutathione, GSMC, GSAC, 49 381 GSPC and especially C393, which gave the most intense signal in the extracted ion 50 382 51 chromatogram of m/z 162.0583. The signal was identified by a combination of not 52 383 identifying any other Scontaining compound at that retention time which might 53 384 plausibly give an insource fragment of m/z 162.0583 and estimation of possible 54 385 retention times under the separation conditions used here with the retention time 55 17 56 386 determined by Yamazaki as described by Block. 57 58 59 60 Metallomics Page 12 of 22 View Article Online DOI: 10.1039/C7MT00098G 1 2 3 387 S1propenylmercaptoglutathione and S2propenylmercaptoglutathione were 4 388 described as Scontaining compounds in garlic first by Nakabayashi et al. 9 but not 5 6 389 quantified. Both compounds are relatives of glutathione containing an additional 7 390 C3H5Sgroup bound to the –SHgroup of glutathione. Bulbs contained more S1 8 391 propenylmercaptoglutathione than S2propenylmercaptoglutathione. In roots the 9 392 concentration of S2propenylmercaptoglutathione was higher than that of S1 10 11 393 propenylmercaptoglutathione (Table S8). Neither compound showed the least 12 394 dependency on Sfertilization levels. 13 14 395 Phytochelatin (PC2, γGluCysγGluCysGly) a pentapeptide often occurring in 15 396 plants was present in root and bulb in its intramolecular oxidised form, showing a 16 17 397 similar behaviour to GSH. The reduced form was not found. PC2 coeluted with 18 398 GSAC(O) compound 1a and was therefore not individually quantified, but its 19 399 concentration in both root and bulb as estimated from the amount of eluting sulphur 20 400 was relative low (Table S11). 21 22 401 23 24 402 Newly identified S-containing compounds in garlic 25 Manuscript 26 27 403 In Fig. 3 the proposed structures for the newly identified compounds in garlic are 28 404 summarised. The extracted ion chromatogram of [M+H]+ 265.0843 (GSMC) showed 29 405 one strong signal (GSMC) and several smaller signals of the same m/z , some of 30 406 + 31 which were insource fragmentation products. One new compound with [M+H] 32 407 265.0843 was identified from its fragmentation pattern as γglutamylhomocysteine 33 408 (γGluHCy). The concentration of γGluHCy in root was Sfertilization rate 34 1 1 409 independent with about 0.25 mmol kg d.w., bulbs contained about 0.07 mmol kg Accepted 35 36 410 d.w. (Table S6, Fig. S16). 37

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 411 Spropylmercaptoglutathione (C 13 H23 N3O6S2), theoretical m/z 381.1028, not yet 39 412 mentioned in the literature was identified by ESIMS/MS, eluting shortly after S2 40 413 propenylmercaptoglutathione. It is coeluting with one of the Sallyl/propenylPC2 41 42 414 isomers. The amount of sulphur eluting at the retention time was low (Fig. S21). 43 415 S1propenylmercaptglutathione and S2propenylmercaptoglutathione both can 44 416 occur, at least theoretically, in their oxidised forms similar to GSAC and GSPC. In 45 417 the case of S1propenylmercaptglutathione and S2propenylmercaptoglutathione 4 46 Metallomics 47 418 different isomers may occur, since the SOgroup can be formed by either of the 48 419 sulphur atoms of molecule (Table S9). These compounds should elute somewhere 49 420 between GSAC(O)/GSPC(O) and their reduced parentcompounds when they 50 421 51 behave similar to GSAC and GSPC. The EICtrace showed both in root and bulb the + 52 422 presence of 4 compounds at [M+H] 396.0894 eluting at about the expected 53 423 retention times. For three of the four compounds ESIMS/MS spectra were 54 424 measured. Compounds 1a and b showed more pronounced fragments at m/z 55 56 425 131.0459 (C 4H7N2O3) and m/z 263.01 (C 8H11 N2O4S2) (Fig. S22 and S23 a and b). 57 426 Compound 2a showed a more pronounced fragment at m/z 120.0126 (C 3H6NO 2S) 58 59 60 Page 13 of 22 Metallomics View Article Online DOI: 10.1039/C7MT00098G 1 2 3 427 (Fig. S23c). The last of the mentioned fragments may indicate that for this compound 4 428 the HSgroup of GSH was oxidised. No further identification with regard to the 5 6 429 influence of double bond position and oxygen position on retention time and 7 430 therefore compound characterisation was possible from the ESIMS/MS data. 8 431 Compound 1a coeluted with oxidised PC2 (Table S13), whereas compounds 1b, 2a 9 432 and 2b eluted without any coeluting Scontaining compounds. Bulbs contained 10 11 433 lower concentrations of the three quantifiable forms than roots (Table S10). Their γ + 12 434 glutamylcysteine counterparts (C 11 H18 N2O5S2 at [M+H] 323.0729 and 13 + 435 C11 H18 N2O6S2 at [M+H] 339.0679) were not detectable. 14 15 436 The, as yet undescribed, Sallyl/propenylcontaining PC2 (C611) was present in 16 17 437 both root and bulb in at least four different isomeric forms (Fig. S25), indicating that 18 438 the Sallyl/propenylgroup can be bound to either SHgroup of PC2. The sulphur 19 439 amount eluting at its retention time was higher than that estimated for PC2 especially 20 440 in bulbs (Table S13). 21 22 441 23 Among the precursor molecules described as part of the biosynthetic pathway by 2 24 442 Block and coworkers one of the first steps during the synthesis of alliin and its

25 443 relatives is the addition of 2methylprop2enoic acid (C 4H6O2) to either γGluCys or Manuscript 26 444 GSH. The products of these two reaction should give molecules with [M+H] + of 27 28 445 337.1064 (2amino5({1carboxy2[(2carboxypropyl)sulfanyl]ethyl}amino)5 29 446 oxopentanoic acid, C336) and [M+H] + of 394.1279 (2amino5({1 30 447 [(carboxymethyl)amino]3[(2carboxypropyl)sulfanyl]1oxopropan2yl}amino)5 31 448 oxopentanoic acid, C393). Not mentioned in the published biosynthetic pathway is 32 + 33 449 the cysteinederivative at [M+H] 208.0638 (2amino3[(2 34 450 carboxypropyl)sulfanyl]propanoic acid, C207). None of these was yet mentioned Accepted 35 451 anywhere else in the literature. C207 and C393 showed strong signals in both ICP 36 37 452 MS/MS and ESIMS in both bulb and root, whereas C336 was not detectable except

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 453 as insource fragment of C393. C207 is the more dominant precursor in root. The 39 454 amount of both compounds was depending on the amount of Sfertilization rate in 40 455 roots but not bulbs (Table S12). Concentrations of C207 and C393 in root were 41 42 456 significantly higher for the highest Sfertilization rate (2 mM S) than in the low and 43 457 medium exposure groups (Table S12). 44 458 45 46 459 Metallomics 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Metallomics Page 14 of 22 View Article Online DOI: 10.1039/C7MT00098G 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 460

25 32 32 Manuscript 26 461 Fig. 1 panel A) Strace from ICPMS/MS; panel B) details of Strace and 27 462 extracted ion chromatogram of m/z 394.1280 of root extract exposed to 2 mM S; 28 463 panel C) ESIMSspectrum of compound; panel D) ESIMS 2spectrum of compound; 29 464 panel E) proposed structure and main fragmentation sites; for HPLCICPMS/ESI 30 465 MS conditions please see Instrumentation; details of all other compounds can be 31 466 found in the electronic supplement. 32 33 467 34 35 Accepted 36 37

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 39 40 41 42 43 44 45 46 Metallomics 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 15 of 22 Metallomics View Article Online DOI: 10.1039/C7MT00098G 1 2 3 4 5 6 7 O

8 16 9 S 32 10 11 32S 12 13 intensityrelative ESIMS C393 14 15 GSAC(O) & 16 GSPC(O) C207 17 18 PC2 19 20

relative intensity ICPMS/MS intensityrelative ICPMS/MS GCMC & y 21 GluHC 22 C379 23 24 0 10 20 30 C611 25 Retention time (min) Manuscript 26 468 27 469 32 28 Fig. 2 Strace from ICPMS/MS and extracted ion chromatograms of 9 sulphur 29 470 containing compounds of root extract exposed to 2 mM S; for HPLCICPMS/ESIMS 30 471 conditions please see Instrumentation; detailed Figures for all compounds can be 31 472 found in the electronic supplement. 32 33 473 34 35 474 Table 2 overview of Scontaining compounds identified in garlic. Accepted 36 37 Compound name Molecular Co ID by Quantified

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. weight / elution MS/MS in literature 39 [M+H] + 40 41 Known compounds in garlic 42 alliin 178.0532 y y 43 Isoaliin 178.0532 y y 44 cycloalliin 178.0532 y y y 45 methiin 152.0376 y y 46 Metallomics 47 propiin 180.0689 y 48 methionine 150.0583 y y 49 methylcysteine 136.0427 y 50 γglutamylSallylcysteine 291.1009 y y 51 γglutamylS1propenylcysteine 291.1009 y y 52 γglutamylS2propenylcysteine 307.0958 y y 53 sulfoxide 54 55 γglutamylS1propenylcysteine 307.0958 y y 56 sulfoxide 57 γglutamylSmethylcysteine 265.0853 y y 58 Glutathione 308.0911 y y y 59 60 Metallomics Page 16 of 22 View Article Online DOI: 10.1039/C7MT00098G 1 2 3 Oxidised glutathione 613.1592 y y 4 Sallylcysteine 162.0583 y 5 S1propenylmercaptoglutathione 380.0944 Y 6 7 S2propenylmercaptoglutathione 380.0944 y 8 9 Newly identified compounds in garlic 10 Phytochelatin 2 538.1272 y y 11 C264 (putative: γGluHCy) 265.0853 y 12 Spropylmercaptoglutathione 382.1105 y y 13 Oxidised forms of S1 396.0894 partly y 14 15 propenylmercaptoglutathione and 16 S2propenylmercaptoglutathione 17 C611 (putative: Sallyl/propenyl 612.1462 partly y 18 containing PC2) 19 C207 (putative: 2amino3[(2 208.0638 y 20 carboxypropyl)sulfanyl]propanoic 21 acid) 22 C336 (putative: 2amino5({1 337.1064 23 24 carboxy2[(2 carboxypropyl)sulfanyl]ethyl}amino) 25 Manuscript 26 5oxopentanoic acid) 27 C393 (putative: 2amino5({1 394.1279 y 28 [(carboxymethyl)amino]3[(2 29 carboxypropyl)sulfanyl]1 30 oxopropan2yl}amino)5 31 oxopentanoic acid) 32 475 33 34 35 Accepted 36 37

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 39 40 41 42 43 44 45 46 Metallomics 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 17 of 22 Metallomics View Article Online DOI: 10.1039/C7MT00098G 1 2 3 4 CH 3 CH 5 2 6 7 O O O O O 8 S O 9 S NH S OH S NH 10 O NH OH 11 O NH 12 HO O HO O 13 NH2 14 NH2 15 Oxidised S-1- and S-2-propenylmercaptoglutathione 16 17 H2C 18 19 20 S 21

22 S S O O O O 23 H 24 NH N HO N NH OH

25 H Manuscript O O NH2 26 O OH 27 S-allyl/propenyl-containing PC-2 28 O 29 30 H3C 31 O OH OH NH O 32 2 O 33 S O S NH H3C OH 34 O NH OH Accepted 35 HO O 36 NH 37 2 38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. Precursor molecules for S-alk(en)ylcysteine sulfoxides 39 O H N 40 2 HS HO O 41 OH

42 NH O O NH 43 HO CH NH S 3 44 O OH S 45 O NH2 O 46 Metallomics 47 γ-Glu-HC S-propylmercaptoglutathione 48 476 49 50 477 Fig. 3 Proposed structures for newly in garlic identified compounds. 51 52 478 53 479 54 55 480 Sulphate 56 481 Inorganic sulphate was eluting from the column in the dead volume. Although by far 57 482 the main component, possible coelution of unknown small sulphur containing 58 59 60 Metallomics Page 18 of 22 View Article Online DOI: 10.1039/C7MT00098G 1 2 3 483 compounds cannot be entirely be excluded. Its concentration in roots was 4 484 depending on the amount of sulphate in the fertilizer, whereas sulphate in bulbs did 5 6 485 not show any dependency on the fertilizer (Table 3, Fig. 4). 7 8 486 Table 3 Mass balance: Sulphate, chromatographically separated unknown 9 487 organic S containing compounds (unk S org ) and chromatographically separated 1 10 488 identified organic Scontaining compounds (known S org ) in mmol S kg d.w. (mean ± 11 489 standard deviation, n = 9 per group), in brackets as average % of total sulphur. 12 13 Sulphate unk S org known S org 14 Root (0.1 mM S) 26 ± 23a,c 78 ± 46 34 ± 14 d 15 (13 %) (39 %) (17 %) 16 Root (0.5 mM S) 63 ± 42 a,b 62 ± 53 45 ± 21e 17 (23 %) (22 %) (16 %) 18 b,c d,e 19 Root (2.0 mM S) 151 ± 40 92 ± 46 140 ± 50 20 (32 %) (20 %) (30 %) 21 Bulb (0.1 7.7 ± 4.9 57 ± 40 32 ± 8.8 22 2 mM S) (5.9 %) (43 %) (24 %) 23 490 b, c : statistically significant difference between groups by Oneway ANOVA p < 0.001 24 491 a: statistically significant difference between groups by Oneway ANOVA p < 0.05 d, e 25 492 : statistically significant difference between groups by Oneway ANOVA p < 0.01 Manuscript 26 493 27 28 494 Overall it is apparent that at lower fertilization rate, the amount of sulphate was lower 29 30 495 and concentration of the identified organosulphur compounds increased, while the 31 496 proportion of unidentified Sspecies decreased (Table 3). 32 33 34 35 Accepted bulb 36 37

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 39 40 root 2 mM S 41 42 43 44 45 root 0.5 mMS 46 Metallomics 47 48 49 root 0.1 mM S 50 51 52 53 0 50 100 150 200 250 300 350 400 54 mM S / kg d.w. 55 unk Sorg sulphate precursors Cys yGluCys yGluCysGly others 56 497 57 58 59 60 Page 19 of 22 Metallomics View Article Online DOI: 10.1039/C7MT00098G 1 2 3 498 Fig. 4 A list of average concentrations (mmol S kg 1) of all Sspecies in garlic roots 4 499 growing under a different Sfertilization regime. Unk Sorg= unknown organic S 5 6 500 containing compounds, precursors = sum of C207 and C393, Cys = sum of methiin, 7 501 alliin, isoalliin, propiin and SAC, γGluCys = sum of GSAC, GSPC, GSAC(O) and 8 502 GSPC(O), γGluCysGly = sum of reduced and oxidised propenylmercapto 9 503 glutathions, others = sum of all other compounds mentioned in the text. 10 11 504 12 13 14 505 Comparing root and bulb sulphur patterns showed that both plant parts were similar 15 506 in their speciation (Fig. S30 & S31) though not necessarily in concentration of the 16 507 individual compounds. The experiments also showed that interplant variability was 17 508 very high with regard to concentrations of Scontaining compounds. The experiments 18 19 509 showed that manipulation of sulphur fertilization rates under hydroponic growth 20 510 conditions did not significantly influence the sulphur content in bulbs . In contrast to 21 511 bulbs the amount of certain Scontaining compounds in roots was strongly 22 512 23 depending on sulphur availability as shown not only by oneway ANOVA, but also by 24 513 PCAanalysis (Fig. 5, Fig. S28 and S29). The main difference was between the

25 514 group exposed to 2 mM sulphur while the 0.1 and 0.5 mM Sfertilized plants were Manuscript 26 515 similar. The excess of S in the hydroponic solution created a significantly different 27 28 516 species distribution of the low molecular weight sulphur species. From the loading 29 517 plot (Fig. S28) it was apparent that the difference were due to the following sulphur 30 518 species: C393, GSAC(O), propiin+GSH+methionin. A smaller influence was exerted 31 519 by Sallyl/propenyl PC2, but not by alliin and isoalliin as was originally expected. 32 33 34 35 PCA, Score plot Accepted 36 4 mM S 37 0.1

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 0.5 3 39 2.0 40 41 2 42 43 1 44

45 PC2 46 0 Metallomics 47 48 -1 49 50 -2 51 52 53 -3 54 -4 -2 0 2 4 6 8 55 PC1 56 520 57 58 521 59 60 Metallomics Page 20 of 22 View Article Online DOI: 10.1039/C7MT00098G 1 2 3 522 Fig. 5 Principal component analysis of all individual plants exposed to different S 4 523 fertilization regime show significant differences in their low molecular weight S 5 6 524 containing species. 7 8 525 9 10 526 Summary 11 527 12 13 528 This has been the first attempted global Sspeciation in a plant. Having the ICP 14 529 MS/MS available for a nontargeted analysis made it possible to determine a full 15 16 530 sulphur mass balance and identified gaps in our knowledge about the multitude of S 17 531 containing compounds in A. sativum . 18 19 532 Using elemental and molecular detectors in parallel (HPLCICPMS/MSESIMS/MS) 20 533 allowed us to identify the molecular composition and assign at least tentative 21 22 534 structures for between 16 and 30 % of the total sulphur. Only a limited number of the 23 535 compounds had been identified and quantified in the literature before due to different 24 536 factors chiefly among them availability of standards, which are necessary when UV

25 Manuscript 537 26 is used for quantification. Among the multitude of identified compounds five not yet 27 538 elsewhere described as occurring in garlic were found and quantified for the first time 28 539 and for a further four the first concentration estimates could be determined. Having 29 540 used a nontargeted Sanalysis a further 20 to 43 % of the total sulphur was 30 31 541 identified as organosulphur compounds but so far no structures and or molecular 32 542 compositions were deducible. 33 34 543 The use of ICPMS/MS in contrast to the use of UVdetection allowed the Accepted 35 544 quantification of all Scontaining compounds independent of whether they were 36 37 545 chromatographically resolved or not without requiring the chromatographic

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 546 separation of every UVabsorbing compound (at the chosen wavelength) and without 39 547 the requirement of speciesspecific standards. Nevertheless quantification of 40 548 compounds on the basis of their molecular weight is still limited in the case of not 41 42 549 fully resolved chromatographic peaks. Hence, chromatography needs to be 43 550 improved if single so far nonresolved compounds need to be quantified in a target 44 551 analysis. 45 46 552 The study also showed, that despite garlic being a wellstudied system, there are still Metallomics 47 48 553 unknown Scontaining compounds present and the use of high resolution ESI 49 554 MS/MS is absolutely required for at least tentative identification of compounds. The 50 555 amount of some of these newly identified compounds was strongly influenced by S 51 556 fertilization. 52 53 557 54 The separation of A. sativum extracts and detection of its sulphur metabolites 55 558 showed also that the chromatographic separation needs to be improved in order to 56 559 quantify the myriad of Scontaining compounds. This sulfomics study by using a non 57 560 target analysis HPLCICPMS/MS/ESIMS demonstrated that all Scontaining 58 59 60 Page 21 of 22 Metallomics View Article Online DOI: 10.1039/C7MT00098G 1 2 3 561 compounds can potentially be detected in plants and it is therefore a useful tool 4 562 when environmental influences on plants are going to be studied. 5 6 563 Acknowledgment: We thank Agilent, UK for access to the Agilent 6200 series 7 8 564 TOF/6500 series QTOF. M.R. especially thanks the ERASMUS programme and G. 9 565 Falasca for support. 10 11 566 12 13 567 14 15 568 16 17 18 1 J. Feldmann, A. Raab, E.M. Krupp, Importance of ICPMS for speciation analysis is 19 changing: future trends for targeted and nontargeted element speciation analysis, 20 Anal. Bioanal. Chem. (2017) DOI 10.1007/s0021601705028 21 2. E. Block, Garlic and other Alliums – The Lore and the Science, Royal Society of 22 23 Chemistry: Cambridge, U.K., 2010. 24 3. A. I. Bhuiyan, V. T. Papajani, M. Paci, S. Melino, Glutathionegarlic sulfur

25 conjugates: slow hydrogen sulfide releasing agents for therapeutic applications, Manuscript 26 Molecules, 2015, 20 , 173150. 27 4. V. Lanzotti, The analysis of onion and garlic , J. Chromatogr. A , 2006, 1112 , 322. 28 5. M. S. Raman, Allicin and Other Functional Active Components in Garlic: Health 29 Benefits and Bioavailability, International Journal of Food Properties , 2007, 10 , 245 30 268. 31 6. E. Block, Fifty years of smelling sulfur , J. Sulfur Chem ., 2013, 34, 158207. 32 33 7. M. Ichikawa, N. Ide, J. Yoshida, H. Yamaguchi, K. Ono, Determination of seven 34 organosulfur compounds in garlic by highperformance liquid chromatography, J. 35 Agric. Food Chem ., 2006, 54 , 153540 . Accepted 36 8. A. Montano, V. M. Beato, F. Mansilla, F. Orgaz, Effect of Genetic Characteristics 37 and Environmental Factors on Organosulfur Compounds in Garlic (Allium sativum L.) 38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. Grown in Andalusia, Spain, J. Agric. Food Chem ., 2011, 59 , 13011307. 39 9. R. Nakabayashi, Y. Sawada, M. Aoyagi, Y. Yamada, M. Y. Hirai, T. Sakurai, T. 40 Kamoi, D. D. Rowan, K. Saito, Chemical Assignment of Structural Isomers of Sulfur 41 Containing Metabolites in Garlic by Liquid ChromatographyFourier Transform Ion 42 43 Cyclotron ResonanceMass Spectrometry, J. Nutr ., 2016, 146 , 397S402S. 44 10. K. O. Amayo, A. H. Petursdottir, C. Newcombe, H. Gunnlaugsdottir, A. Raab, E. 45 Krupp, J. Feldmann, Identification and Quantification of Arsenolipids Using 46 ReversedPhase HPLC Coupled Simultaneously to HighResolution ICPMS and Metallomics 47 HighResolution Electrospray MS without SpeciesSpecific Standards, Anal. Chem., 48 2011, 83, 35893595. 49 11. K. Bluemlein, A. Raab, J. Feldmann, Stability of arsenic peptides in plant 50 extracts: offline versus online parallel elemental and molecular mass spectrometric 51 detection for liquid chromatographic separation, Anal. Bioanal. Chem ., 2009, 393 , 52 53 357366. 54 12. http://msbi.ipbhalle.de/MetFusion/ 55 13. http://www.chemspider.com/ 56 14. K. Ghasemi, S. Bolandnazar, S. J.Tabatabaei, H. Pirdashti, M. Arzanlou, M. A. 57 Ebrahimzadeh, H. Fathi, Antioxidant properties of garlic as affected by selenium and 58 59 60 Metallomics Page 22 of 22 View Article Online DOI: 10.1039/C7MT00098G 1 2 3 4 humic acid treatments , New Zealand Journal Of Crop And Horticultural Science , 5 2015, 43 , 173181. 6 15. E. Bloem, S. Haneklaus, E. Schnug, Influence of fertilizer practices on S 7 containing metabolites in garlic (Allium sativum L.) under field conditions, J. Agric. 8 9 Food Chem., 2012, 58 , 1069010696. 10 16. M. Yoo, S. Lee, S. Kim, J. B. Hwang, J. Choe, D. Shin, Composition of 11 organosulfur compounds from cool and warmtype garlic (Allium sativum L.) in 12 Korea, Food Sci. Biotech. , 2014, 23 , 337344. 13 17. Y. Yamazaki, T. Tokunaga, T. Okuno, Quantitative determination of eleven flavor 14 precursors (Salk(en)yl cysteine derivatives) in garlic with HPLC method, Nippon 15 Shokuhin Kagaku Kogaku Kaishi, 2005, 52, 160166; in E. Block, Garlic and other 16 Alliums – The Lore and the Science, Royal Society of Chemistry: Cambridge, U.K., 17 2010. 18 19 18. J. Hughes, H. A. Collin, A. Tregova, A. B. Tomsett, R. Cosstick, M. G. Jones, 20 Effect of low storage temperature on some of the flavour precursors in garlic (Allium 21 sativum), Plant Foods Hum. Nutr ., 2006, 61 , 8185. 22 23 24

25 Manuscript 26 27 28 29 30 31 32 33 34 35 Accepted 36 37

38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 39 40 41 42 43 44 45 46 Metallomics 47 48 49 50 51 52 53 54 55 56 57 58 59 60